F , Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel . The average intensity at the front and the back is normalized to the intensity at the sides , demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation . Error bars correspond to the standard deviation . Constriction : n = 17 cells over 4 biological replicates , 15 µm channels : n = 20 cells over 3 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


G , GFP - nesprin - 2 cells deformed by the pressure gradient in a micropipette aspiration device . Note that the GFP signal does not increase at the tip but increases at sides of the nucleus .

O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O

G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.


A , CRISPR - modified cells ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) and mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction . B , Quantification of intensity of GFP - nesprin - 2 and mCh - LAC , normalized to the intensity at the sides . Error bars correspond to the standard deviation . Constriction : n = 19 cells over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility . Inset are shown close - up views of the bleached areas at the front of the nucleus . B , Quantification of the recovery . Error bars correspond to the standard error of the mean , n = 24 over 3 biological replicates .

O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.


A , Expression of the artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in cells migrating through narrow constrictions .

O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions.


C , Quantification of the mini - nesprin - 2G accumulation of cells migrating through narrow constrictions . Note that the mutant construct does not accumulate at the front of the nucleus . Error bars correspond to the standard deviation . Mini - nesprin - 2G : n = 10 cells over 4 biological replicates ; Mutant : n = 11 cells over 4 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


A , Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces . Cross - sections along the direction of migration ( yellow ) and perpendicular to the direction of migration ( blue , 1 - 4 ) are shown below .

O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O

A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.


B , A confocal slice on the surface of the nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at the surface of the nucleus . Dashed white lines represent the outlines of the constriction .

O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O

B, A confocal slice on the surface of the nucleus indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.


C , Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope . Dashed white lines represent the outlines of the constriction . The distance between mCh - LAC and GFP - nesprin - 2 was measured at the back , at the constriction , at the front near the constriction , and at the center of the front . ( N = 17 cells , over 3 biological replicates . The alignment of the two channels was corrected using at least 28 fluorescent beads . )

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)


D , Two - photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus , but not ablation at the back . The nuclei of DN - KASH , which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei . Bars represent the average value . Error bars represent the standard error on the mean . Back : N = 9 , Front : N = 30 , Cell Front : N = 16 , and DNKASH : N = 31 , 3 biological replicates .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.


( A ) BMDMs from WT ( Pml + / + ) and Pml - / - mice were pre - treated with zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 30 min followed by co - treatment with AT - 406 ( 1 μM ) . Cell viability was evaluated by ATP release after 18 h . Data show mean ± SD of four biological replicates . ( B ) WT and Pml - / - BMDM were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) and Nec - 1 ( 40 μM ) or GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates .

O O O B-CELL_TYPE O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(A) BMDMs from WT (Pml+/+) and Pml-/- mice were pre-treated with zVAD (20 μM), Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 30 min followed by co-treatment with AT-406 (1 μM). Cell viability was evaluated by ATP release after 18 h. Data show mean ± SD of four biological replicates. (B) WT and Pml-/- BMDM were treated with TNF (100 ng/ml), zVAD (20 μM) and Nec-1 (40 μM) or GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates.


( D ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) , and GSK872 ( 10 μM ) , as indicated , for 8 h . Cell viability was determined by ATP assay . Data show mean ± SD of three biological replicates . ( E ) WT and Pml - / - immortalized MEFs were treated with TNF ( 100 ng / ml ) , IKK inhibitor BMS - 345541 ( 10 mM ) and Nec - 1 , as indicated , for 6 h and then cell death was assayed . Data show mean ± SD of five biological replicates , except the Nec - 1 - containing experiment ( n = 2 ) .

O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).


( A , B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) ( A ) or TNF + zVAD ( Tz ) ( B ) , as in Fig . 1 , for the indicated times . Contents of RIPK1 , p - RIPK1 [ S166 ] , RIPK3 , p - RIPK3 , MLKL , and p - MLKL in cell lysates was analysed by immunoblots . Bottom panels , quantitation of p - RIPK1 [ S166 ] from three biological replicates using normalized intensity of p - RIPK1 [ S166 ] in WT cells at 4 h as 1 . Data show mean ± SD .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

(A, B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) (A) or TNF + zVAD (Tz) (B), as in Fig. 1, for the indicated times. Contents of RIPK1, p-RIPK1[S166], RIPK3, p-RIPK3, MLKL, and p-MLKL in cell lysates was analysed by immunoblots. Bottom panels, quantitation of p-RIPK1[S166] from three biological replicates using normalized intensity of p-RIPK1[S166] in WT cells at 4 h as 1. Data show mean ± SD.


( D ) BMDMs were treated with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) for 18 h , and TNF generated was then analyzed by ELISA . Data show mean ± SD of four biological replicates . ( E ) BMDMs were treated with zVAD + AT - 406 ( 1 μM ) , with or without anti - TNF for 18 h , and cell viability quantitated . Data show mean ± SD of three biological replicates .

O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(D) BMDMs were treated with zVAD (20 μM) +AT-406 (1 μM) for 18 h, and TNF generated was then analyzed by ELISA. Data show mean ± SD of four biological replicates. (E) BMDMs were treated with zVAD + AT-406 (1 μM), with or without anti-TNF for 18 h, and cell viability quantitated. Data show mean ± SD of three biological replicates.


( A ) Pml + / + and Pml - / - BMDMs were treated with zVAD for 30 min , followed by AT - 406 ( zA ) treatment at the indicated time - points , before preparing total cell lysates ( TCL ) . TCL were immunoprecipitated with anti - FADD and the amounts of p - RIPK1 [ S166 ] , RIPK1 and caspase - 8 in the precipitates and TCL were analysed . ( B ) Pml + / + and Pml - / - BMDMs were treated with zVAD + AT406 ( zA ) as in ( A ) , and TCL were then harvested . TCL were immunoprecipitated with anti - caspase - 8 , and the contents of p - RIPK1 [ S166 ] and RIPK1 in the precipitates and TCL was analysed .

O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

(A) Pml+/+ and Pml-/- BMDMs were treated with zVAD for 30 min, followed by AT-406 (zA) treatment at the indicated time-points, before preparing total cell lysates (TCL). TCL were immunoprecipitated with anti-FADD and the amounts of p-RIPK1[S166], RIPK1 and caspase-8 in the precipitates and TCL were analysed. (B) Pml+/+ and Pml-/- BMDMs were treated with zVAD + AT406 (zA) as in (A), and TCL were then harvested. TCL were immunoprecipitated with anti-caspase-8, and the contents of p-RIPK1[S166] and RIPK1 in the precipitates and TCL was analysed.


( A , B ) WT ( Pml + / + ) and Pml - / - mice ( n = 12 in each group ) were injected with mouse TNF ( 1 . 5 μg / g body weight ) , and rectal body temperature ( A ) and survival ( B ) were determined at the indicated time - points . # , the temperature of only six mice was measured at the indicated time - points . Mean ± S . E . M is shown . * P < 0 . 05 as determined by two - way ANOVA ( A ) , or by Log - rank ( Mantel - Cox ) test ( B ) .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) WT (Pml+/+) and Pml-/- mice (n = 12 in each group) were injected with mouse TNF (1.5 μg/g body weight), and rectal body temperature (A) and survival (B) were determined at the indicated time-points. #, the temperature of only six mice was measured at the indicated time-points. Mean ± S.E.M is shown. *P < 0.05 as determined by two-way ANOVA (A), or by Log-rank (Mantel-Cox) test (B).


( A ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated timeframes . The contents of p - IKKα / β , IKKα / β , IκBα , p - MK2 and MK2 was analysed by immunoblotting . ( B ) Pml + / + and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times , and levels of p - RIPK1 [ S321 ] , RIPK1 , p - JNK , JNK , p - ERK , ERK , p - p38 , and p38 at the indicated time points were then determined .

O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O

(A) Pml+/+ and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated timeframes. The contents of p-IKKα/β, IKKα/β, IκBα, p-MK2 and MK2 was analysed by immunoblotting. (B) Pml+/+and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times, and levels of p-RIPK1[S321], RIPK1, p-JNK, JNK, p-ERK, ERK, p-p38, and p38 at the indicated time points were then determined.


( C , D , E , F ) WT ( Pml + / + ) and Pml - / - mice were treated with DMSO or Nec - 1s ( 6 μg / g ) or MK2 inhibitor PF - 3644022 ( 75 μg / mouse ) by intraperitoneal injection 15 min before and 60 min after intravenous TNF ( 1 . 5 μg / g body weight ) injection . Body temperature ( C , E ) and survival ( D , F ) were monitored . Experiments C - F were conducted concurrently . Mean ± S . E . M is shown ( C , E ) .

O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D, E, F) WT (Pml+/+) and Pml-/- mice were treated with DMSO or Nec-1s (6 μg/g) or MK2 inhibitor PF-3644022 (75 μg/mouse) by intraperitoneal injection 15 min before and 60 min after intravenous TNF (1.5 μg/g body weight) injection. Body temperature (C, E) and survival (D, F) were monitored. Experiments C-F were conducted concurrently. Mean ± S.E.M is shown (C, E).


( A , B ) WT and Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) ( A ) or with zVAD ( 20 μM ) + AT - 406 ( 1 μM ) ( B ) , and then the contents of p - RIPK1 [ S321 ] , RIPK1 , p - p38 , p38 was determined at the indicated time - points .

O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(A, B) WT and Mk2-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) (A) or with zVAD (20 μM) + AT-406 (1 μM) (B), and then the contents of p-RIPK1[S321], RIPK1, p-p38, p38 was determined at the indicated time-points.


( C ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with zVAD ( 20 μM ) , AT - 406 ( 1 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 18 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method . ( D ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - BMDMs were treated with TNF ( 100 ng / ml ) , zVAD ( 20 μM ) , Nec - 1 ( 40 μM ) and GSK872 ( 10 μM ) , as indicated , for 16 h . Cell viability was determined by ATP release . Data show mean ± SD of three biological replicates . * P < 0 . 05 for multiple t - tests according to the Holm - Sidak method .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with zVAD (20 μM), AT-406 (1 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 18 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method. (D) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- BMDMs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM) and GSK872 (10 μM), as indicated, for 16 h. Cell viability was determined by ATP release. Data show mean ± SD of three biological replicates. *P < 0.05 for multiple t-tests according to the Holm-Sidak method.


( F ) WT , Pml - / - , Mk2 - / - and Pml - / - Mk2 - / - mice were injected i . v . with TNF ( 1 . 5 mg / kg ) , and survival was monitored for 24 h . Data represent a combination of two independent experiments ( n = 10 ) . * P < 0 . 05 for Log - rank ( Mantel - Cox ) test .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) WT, Pml-/-, Mk2-/- and Pml-/-Mk2 -/- mice were injected i.v. with TNF (1.5 mg/kg), and survival was monitored for 24 h. Data represent a combination of two independent experiments (n=10). *P < 0.05 for Log-rank (Mantel-Cox) test.


( D ) WT and Pml - / - BMDMs were treated with TNF ( 100 ng / ml ) + zVAD ( 20 μM ) for the indicated times . p38 MAPK was immunoprecipitated , and its association with MK2 was then analysed .

O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O O O

(D) WT and Pml-/- BMDMs were treated with TNF (100 ng/ml) + zVAD (20 μM) for the indicated times. p38 MAPK was immunoprecipitated, and its association with MK2 was then analysed.


( E ) Recombinant Flag - PML - I , GST - MK2 ( 60 ng ) and His - p38 ( 60 ng ) were incubated , as indicated , at 4 ° C for 2 h . p38 was pulled down by anti - p38 and then PML , MK2 and p38 contents in the precipitate and reaction mixture ( input ) were detected by immunoblotting .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O

(E) Recombinant Flag-PML-I, GST-MK2 (60 ng) and His-p38 (60 ng) were incubated, as indicated, at 4 °C for 2 h. p38 was pulled down by anti-p38 and then PML, MK2 and p38 contents in the precipitate and reaction mixture (input) were detected by immunoblotting.


( F ) Recombinant Flag - PML - I , GST - MK2 ( 100 ng ) and His - p38 ( 200 ng ) were incubated , as indicated , at 30 ° C in in vitro kinase assay buffer ( 20 mM HEPES , 10 mM MgCl2 , 100 nM ATP , 1 mM DTT ) for 20 min . The levels of p - MK2 , MK2 , p38 , and PML ( FLAG ) in the reaction mixture were then determined .

O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O

(F) Recombinant Flag-PML-I, GST-MK2 (100 ng) and His-p38 (200 ng) were incubated, as indicated, at 30 °C in in vitro kinase assay buffer (20 mM HEPES, 10 mM MgCl2, 100 nM ATP, 1 mM DTT) for 20 min. The levels of p-MK2, MK2, p38, and PML (FLAG) in the reaction mixture were then determined.


( A ) mH2A1 . 2 and different neurogenesis markers are present in cortical lysates collected at various time points ( E13 , E15 , E17 , and P0 ) .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O

(A) mH2A1.2 and different neurogenesis markers are present in cortical lysates collected at various time points (E13, E15, E17, and P0).


( C ) A scatter diagram shows the change trend of mH2A1 . 2 and different neurogenesis markers . n = 5 mice , independent replicates .

O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O O

(C) A scatter diagram shows the change trend of mH2A1.2 and different neurogenesis markers. n = 5 mice, independent replicates.


( E ) Electroporation of mH2A1 . 2 - shRNA results in abnormal cell distribution in the developing neocortex . Electroporation was performed at E13 . 5 , and the mouse was harvested at E16 . 5 . Scale bar represents 50 μm . ( F ) Graph shows the percentage of GFP - positive cells distributed in the VZ / SVZ , IZ and CP . n = 7 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Electroporation of mH2A1.2-shRNA results in abnormal cell distribution in the developing neocortex. Electroporation was performed at E13.5, and the mouse was harvested at E16.5. Scale bar represents 50 μm. (F) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 7 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.01(**) or P < 0.001(***). n.s., not significant.


( D ) Abnormal cell distribution was observed in the mH2A1 . 2 silenced neocortex . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( E ) Graphs of the percentage of GFP - positive cells in the VZ / SVZ , IZ and CP . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D)Abnormal cell distribution was observed in the mH2A1.2 silenced neocortex. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (E) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( F ) Brain sections of WT and KO mice at E16 . 5 were immunostained with the mitotic marker pH3 and DAPI . Scale bar represents 20 μm . ( G ) Statistics of pH3 + cells of the cortex . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O B-TISSUE O O O O O B-ORGANISM O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Brain sections of WT and KO mice at E16.5 were immunostained with the mitotic marker pH3 and DAPI. Scale bar represents 20 μm. (G) Statistics of pH3+ cells of the cortex. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( H ) Representative images of E16 . 5 coronal brain sections were immunostained for PAX6 . The GFP plasmid was electroporated into WT and KO mice brains at E13 . 5 , and the mice were sacrificed at E16 . 5 . Scale bar represents 50 μm . ( I ) Quantification of GFP and Pax6 double positive cells in the VZ / SVZ . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant .

O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative images of E16.5 coronal brain sections were immunostained for PAX6. The GFP plasmid was electroporated into WT and KO mice brains at E13.5, and the mice were sacrificed at E16.5. Scale bar represents 50 μm. (I) Quantification of GFP and Pax6 double positive cells in the VZ/SVZ. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**) or P < 0.001(***). n.s., not significant.


( A ) Representative images of E16 . 5 coronal brain sections immune stained for Tbr2 and BrdU . BrdU was injected intraperitoneally into pregnant mice at E16 . 5 for 2h of pulse labeling . Scale bar represents 20 μm . ( B ) Statistics of TBR2 + cells per 100μm2 surface of VZ / SVZ . The number of TBR2 + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . ( C ) Statistics of BrdU + cells per 100μm2 surface of VZ / SVZ . The number of BrdU + cells increased significantly in KO mice compared to WT mice . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O O O O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-TISSUE O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of E16.5 coronal brain sections immune stained for Tbr2 and BrdU. BrdU was injected intraperitoneally into pregnant mice at E16.5 for 2h of pulse labeling. Scale bar represents 20 μm. (B) Statistics of TBR2+ cells per 100μm2 surface of VZ/SVZ. The number of TBR2+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. (C) Statistics of BrdU+ cells per 100μm2 surface of VZ/SVZ. The number of BrdU+ cells increased significantly in KO mice compared to WT mice. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( E ) Cell cycle exit was decreased in KO mice . E16 . 5 brain sections were stained with anti - BrdU and anti - Ki67 in WT mice and KO mice that were administered BrdU ( 100 mg / kg ) for 24 h and harvested at E16 . 5 . Scale bar represents 20 μm . ( F ) Percentage of cell cycle exit ( BrdU + Ki67 − / BrdU + ) in WT mice and KO mice . n = 7 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O O O B-TISSUE O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-ORGANISM O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-ORGANISM O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Cell cycle exit was decreased in KO mice. E16.5 brain sections were stained with anti-BrdU and anti-Ki67 in WT mice and KO mice that were administered BrdU (100 mg/kg) for 24 h and harvested at E16.5. Scale bar represents 20 μm. (F) Percentage of cell cycle exit (BrdU+Ki67−/ BrdU+) in WT mice and KO mice. n=7 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( G ) Western blot analysis showed that the protein expression levels of neural stem cell marker , including TBR2 , PCNA , and PAX6 are increased in mH2A1 . 2 KO mice compared with WT . ( H ) Statistics of the normalized density of TBR2 , PCNA , and PAX6 . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G)Western blot analysis showed that the protein expression levels of neural stem cell marker, including TBR2, PCNA, and PAX6 are increased in mH2A1.2 KO mice compared with WT. (H) Statistics of the normalized density of TBR2, PCNA, and PAX6. n=5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( K ) Coronal brain slices of E16 . 5 WT mice and KO mice were immunostained using anti - TUJ1 . Scale bar represents 20 μm . ( L ) Relative TUJ1 + cells in the VZ of WT mice and KO mice . TUJ1 was reduced in cortical . n = 6 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O B-TISSUE O O O O O O B-ORGANISM O O B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O B-ORGANISM O O B-ORGANISM O B-GENEPROD O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Coronal brain slices of E16.5 WT mice and KO mice were immunostained using anti-TUJ1. Scale bar represents 20 μm. (L) Relative TUJ1+ cells in the VZ of WT mice and KO mice. TUJ1 was reduced in cortical. n=6 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( M ) The apoptosis levels between WT and KO brains showed no significant difference . E16 . 5 brain sections of WT and KO mice were subjected to TUNEL assay . Scale bar represents 50 μm . ( N ) Graph shows the numbers of apoptotic cells per field . n = 4mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) . n . s . , not significant .

O O O O B-EXP_ASSAY O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) The apoptosis levels between WT and KO brains showed no significant difference. E16.5 brain sections of WT and KO mice were subjected to TUNEL assay. Scale bar represents 50 μm. (N) Graph shows the numbers of apoptotic cells per field. n=4mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*). n.s., not significant.


( A ) Representative images of neurons after 4 days of culture in vitro . GFP expressing plasmid was electroporated into the E13 . 5 cerebral cortices of WT and KO mice to label neural progenitor cells . After 24 h , the GFP + cells were isolated and cultured in differentiation medium for 4 days . Scale bar represents 15 μm . ( B ) Graph shows that the total dendritic length of neurons is decreased when mH2A1 . 2 is deleted . n = 25 cells from four samples . ( C ) Statistics show that the number of branch points is reduced upon mH2A1 . 2 deletion . n = 25 cells from four samples . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) .

O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of neurons after 4 days of culture in vitro. GFP expressing plasmid was electroporated into the E13.5 cerebral cortices of WT and KO mice to label neural progenitor cells. After 24 h, the GFP+ cells were isolated and cultured in differentiation medium for 4 days. Scale bar represents 15 μm. (B) Graph shows that the total dendritic length of neurons is decreased when mH2A1.2 is deleted. n = 25 cells from four samples. (C) Statistics show that the number of branch points is reduced upon mH2A1.2 deletion. n = 25 cells from four samples. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(**).


( F ) The brain sections were immunostained with CUX1 and CTIP2 ( marker of layer I ; marker of layer V ) at P0 . Scale bar represents 20 μm . n = 4 mice , independent replicates . ( G ) Statistics of CUX1 + cells in the upper layer . CUX1 + cells were increased in KO mice n = 4 mice , independent replicates . ( H ) Statistics of CTIP2 + cells in the deep layer . CTIP2 + cells were reduced in KO mice . n = 4 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 (

O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The brain sections were immunostained with CUX1 and CTIP2 (marker of layer I; marker of layer V) at P0. Scale bar represents 20 μm. n=4 mice, independent replicates. (G) Statistics of CUX1+cells in the upper layer. CUX1+cells were increased in KO mice n=4 mice, independent replicates. (H)Statistics of CTIP2+ cells in the deep layer. CTIP2+ cells were reduced in KO mice. n=4 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05(*), P < 0.01(


( A ) Representative tracing path of WT mice and KO mice in the open field test . ( B ) The total traveling distance was not different between WT and KO mice . ( C ) KO mice spent less time in the center than WT mice . Date information : All mice were male , 8 - 12 weeks old . WT , n = 12 mice ; KO , n = 12 mice . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) or P < 0 . 001 ( * * * ) . n . s . , not significant . Analysis was performed by researcher blinded to the experimental conditions .

O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative tracing path of WT mice and KO mice in the open field test. (B) The total traveling distance was not different between WT and KO mice. (C) KO mice spent less time in the center than WT mice. Date information: All mice were male, 8-12 weeks old. WT, n=12 mice; KO, n=12 mice. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P< 0.001(***). n.s., not significant. Analysis was performed by researcher blinded to the experimental conditions.


( E ) Graph shows the percentage of GFP + cells in the VZ / SVZ , IZ and CP . The number of GFP + cells in the VZ / SVZ region increased significantly , and the number of GFP + in the CP layer decreased significantly . n = 5 mice , independent replicates . Date information : Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Graph shows the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. The number of GFP+ cells in the VZ/SVZ region increased significantly, and the number of GFP+ in the CP layer decreased significantly. n =5 mice, independent replicates. Date information: Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


( I ) Representative images of E16 . 5 cortices that had been electroporated with GFP or GFP + NKX2 . 2 into WT and KO brains at E13 . 5 . Scale bar represents 50 μm . ( J ) Graphs of the percentage of GFP + cells in the VZ / SVZ , IZ and CP . Overexpression of NKX2 - 2 restored the distribution of GFP + cells to the normal state . n = 5 mice , independent replicates . Date information : Representative images from at least three independent experiments . Error bars represent the means ± S . E . M . ; Two - tailed unpaired t - test , P < 0 . 05 ( * ) , P < 0 . 01 ( * * ) . n . s . , not significant .

O O O O O O O O O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Representative images of E16.5 cortices that had been electroporated with GFP or GFP+NKX2.2 into WT and KO brains at E13.5. Scale bar represents 50 μm. (J) Graphs of the percentage of GFP+ cells in the VZ/SVZ, IZ and CP. Overexpression of NKX2-2 restored the distribution of GFP+ cells to the normal state. n=5 mice, independent replicates. Date information: Representative images from at least three independent experiments. Error bars represent the means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**). n.s., not significant.


A ) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex , anterior cingulate cortex , premotor cortex , lateral temporal cortex , and hippocampus . The heatmap indicates the associated T - statistic for each voxel , with light blue representing the highest value .

O O B-DISEASE O O O O O B-EXP_ASSAY O O O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O

A) ALS patients from the UPenn Biobank neuroimaging cohort with higher wPRS exhibited greater reduction of cortical thickness in the orbital prefrontal cortex, anterior cingulate cortex, premotor cortex, lateral temporal cortex, and hippocampus. The heatmap indicates the associated T-statistic for each voxel, with light blue representing the highest value.


Sites that bound either HIF - 1α ( A & B ) or HIF - 2α ( C & D ) in both replicates were identified by the MACS peak caller and ordered on the y - axis according to HIF - 1β signal intensity . Heatmaps show HIF ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity , averaged across two independent ChIP - seq experiments ) at HIF binding sites and across the flanking ± 5kb regions ( x - axis ) . ( A ) HIF - 1β signal intensity and ( B ) HIF - 1α signal intensity at HIF - 1α binding sites and ( C ) HIF - 1β signal intensity and ( D ) HIF - 2α signal intensity at HIF - 2α binding sites . HIF - 1β signal intensity above local background levels is observed at all HIF - 1α and HIF - 2α binding sites . ( E & F ) Line plots showing average ( n = 2 , independent ChIP - seq experiments ) HIF - 1β signal intensity ( solid blue lines ) within the MACS defined ( E ) HIF - 1α and ( F ) HIF - 2α binding sites compared to the average background HIF - 1β binding signal ( dashed black line ) at non - HIF - α - binding accessible sites ( defined by FAIRE - seq ) . Sites are ranked on the x - axis according to HIF - 1β signal . HIF - 1β signal intensity at both HIF - 1α and HIF - 2α binding sites was consistently above genome - wide background levels . ( G ) HIF - 1β signal intensity was plotted against total HIF - α ( HIF - 1α + HIF - 2α ) signal intensity for all sites that bound one or more HIF subunits . Strong correlation was observed between HIF - α and HIF - 1β signal intensities . ( H ) The ratio of total HIF - α signal to HIF - 1β signal ( x - axis ) was determined for each site and plotted as a frequency distribution ( y - axis ) showing a tight unimodal distribution . ( I ) The non - reproducibility of the total HIF - α / HIF - 1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate

O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

Sites that bound either HIF-1α (A & B) or HIF-2α (C&D) in both replicates were identified by the MACS peak caller and ordered on the y-axis according to HIF-1β signal intensity. Heatmaps show HIF ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity, averaged across two independent ChIP-seq experiments) at HIF binding sites and across the flanking ±5kb regions (x-axis). (A) HIF-1β signal intensity and (B) HIF-1α signal intensity at HIF-1α binding sites and (C) HIF-1β signal intensity and (D) HIF-2α signal intensity at HIF-2α binding sites. HIF-1β signal intensity above local background levels is observed at all HIF-1α and HIF-2α binding sites. (E & F) Line plots showing average (n = 2, independent ChIP-seq experiments) HIF-1β signal intensity (solid blue lines) within the MACS defined (E) HIF-1α and (F) HIF-2α binding sites compared to the average background HIF-1β binding signal (dashed black line) at non-HIF-α-binding accessible sites (defined by FAIRE-seq). Sites are ranked on the x-axis according to HIF-1β signal. HIF-1β signal intensity at both HIF-1α and HIF-2α binding sites was consistently above genome-wide background levels. (G) HIF-1β signal intensity was plotted against total HIF-α (HIF-1α + HIF-2α) signal intensity for all sites that bound one or more HIF subunits. Strong correlation was observed between HIF-α and HIF-1β signal intensities. (H) The ratio of total HIF-α signal to HIF-1β signal (x-axis) was determined for each site and plotted as a frequency distribution (y-axis) showing a tight unimodal distribution. (I) The non-reproducibility of the total HIF-α/HIF-1β ratio was assessed by plotting the ratio in replicate 1 versus that in replicate 2


For each cell line , genes were ranked according to hypoxic induction ( 0 . 5 % hypoxia for 16 hours ) in RNA - seq analyses ( n = 3 ) . Enrichment of genes ( TSS ) closest to each canonical HIF - α / β binding site ( i . e . identified in both HIF - 1α ( or HIF - 2α ) and both HIF - 1β ChIP - seq experiments ) was then examined using Gene Set Enrichment Analysis ( ES = enrichment score , NES = normalized enrichment score )

O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

For each cell line, genes were ranked according to hypoxic induction (0.5% hypoxia for 16 hours) in RNA-seq analyses (n = 3). Enrichment of genes (TSS) closest to each canonical HIF-α/β binding site (i.e. identified in both HIF-1α (or HIF-2α) and both HIF-1β ChIP-seq experiments) was then examined using Gene Set Enrichment Analysis (ES = enrichment score, NES = normalized enrichment score)


Immunoblots show progressive induction of HIF - 1α and HIF - 2α protein levels with increasing severity of

B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O

B-EXP_ASSAY 0 0 0 0 B-GENEPROD I-GENEPROD I-GENEPROD 0 B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY 0 0 0 0

Immunoblots show progressive induction of HIF-1α and HIF-2α protein levels with increasing severity of hypoxia


HIF - 1α ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O

HIF-1α ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 0 . 5 % hypoxia is plotted against that at 3 % hypoxia for all canonical HIF - binding sites that bound at one oxygen concentration or the other or both . ChIP - seq signal was increased at 0 . 5 % oxygen , but correlated well with that at 3 % oxygen and no new sites were

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O

(C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 0.5% hypoxia is plotted against that at 3% hypoxia for all canonical HIF-binding sites that bound at one oxygen concentration or the other or both. ChIP-seq signal was increased at 0.5% oxygen, but correlated well with that at 3% oxygen and no new sites were generated


Frequency distribution of ChIP - seq signals at 0 . 5 % oxygen compared to 3 % oxygen . The ratio of total HIF - α signal was unimodally distributed ( purple line ) . Upper tertile HIF - α sites ( solid red line ) had a significantly ( p = 10 - 7 , Wilcox rank sum test ) higher ratio of HIF - 1β signal ( dotted red line ) and vice versa ( blue lines )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

Frequency distribution of ChIP-seq signals at 0.5% oxygen compared to 3% oxygen. The ratio of total HIF-α signal was unimodally distributed (purple line). Upper tertile HIF-α sites (solid red line) had a significantly (p = 10-7, Wilcox rank sum test) higher ratio of HIF-1β signal (dotted red line) and vice versa (blue lines)


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at 0 . 5 % oxygen was plotted against that at 3 % oxygen . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the two oxygen

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at 0.5% oxygen was plotted against that at 3% oxygen. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the two oxygen concentrations


Immunoblots show induction of HIF - 1α and HIF - 2α protein level , which is highest at 6 hours and falls by 48

B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

Immunoblots show induction of HIF-1α and HIF-2α protein level, which is highest at 6 hours and falls by 48 hours


HIF - 1α , ( C ) HIF - 2α and ( D ) HIF - 1β ChIP - seq signal intensity ( averaged across two independent ChIP - seq experiments ) at 16 hours is plotted against that at 6 hours for all canonical HIF - binding sites that bound at any of the three timepoints . ChIP - seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia , but correlated well between the two timepoints and no novel sites were observed at either timepoint . ( E - F ) The same plots comparing ( E ) HIF - 1α , ( F ) HIF - 2α and ( G ) HIF - 1β ChIP - seq signals at 48 hours and 6 hours hypoxia . ChIP - seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either

B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

HIF-1α, (C) HIF-2α and (D) HIF-1β ChIP-seq signal intensity (averaged across two independent ChIP-seq experiments) at 16 hours is plotted against that at 6 hours for all canonical HIF-binding sites that bound at any of the three timepoints. ChIP-seq signal was increased at 16 hours hypoxia compared to 6 hours hypoxia, but correlated well between the two timepoints and no novel sites were observed at either timepoint. (E-F) The same plots comparing (E) HIF-1α, (F) HIF-2α and (G) HIF-1β ChIP-seq signals at 48 hours and 6 hours hypoxia. ChIP-seq signal at 48 hours correlated well with that at 6 hours and again no novel sites were observed at either timepoint


Frequency distribution of ChIP - seq signals at 48 hours compared to 6 hours hypoxia . The ratio of total HIF - α signal had a unimodal distribution ( purple line ) . However , upper tertile HIF - α sites ( solid red line ) had a comparable ratio of HIF - 1β signal ( dotted red line ) to the ratio of HIF - 1β signal in the lower tertile HIF - α sites ( blue lines ) suggesting that differences in HIF - α signal resulted from random variation rather than a true biological

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

Frequency distribution of ChIP-seq signals at 48 hours compared to 6 hours hypoxia. The ratio of total HIF-α signal had a unimodal distribution (purple line). However, upper tertile HIF-α sites (solid red line) had a comparable ratio of HIF-1β signal (dotted red line) to the ratio of HIF-1β signal in the lower tertile HIF-α sites (blue lines) suggesting that differences in HIF-α signal resulted from random variation rather than a true biological difference


The ratio of HIF - 1α to HIF - 2α ChIP - seq signal for all canonical HIF - binding sites at ( I ) 16 hours hypoxia and at ( J ) 48 hours hypoxia was plotted against that at 6 hours hypoxia . Strong correlation in the HIF - 1α to HIF - 2α ratio was observed between the various

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

The ratio of HIF-1α to HIF-2α ChIP-seq signal for all canonical HIF-binding sites at (I) 16 hours hypoxia and at (J) 48 hours hypoxia was plotted against that at 6 hours hypoxia. Strong correlation in the HIF-1α to HIF-2α ratio was observed between the various timepoints


Cells were incubated in 0 . 5 % ambient oxygen for 16 hours . Immunoblots show HIF - 1α , HIF - 2α and HIF - 1β protein levels in the wild type and single clones of CRISPR - Cas9 engineered cell

O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O

Cells were incubated in 0.5% ambient oxygen for 16 hours. Immunoblots show HIF-1α, HIF-2α and HIF-1β protein levels in the wild type and single clones of CRISPR-Cas9 engineered cell lines


HIF - 1α ChIP - seq signal in the HIF - 2α KO cells was plotted against that in the wild - type cells for all canonical HIF - 1 binding sites in either cell

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

HIF-1α ChIP-seq signal in the HIF-2α KO cells was plotted against that in the wild-type cells for all canonical HIF-1 binding sites in either cell type


HIF - 2α ChIP - seq signal in the HIF - 1α KO cells was plotted against that in the wild - type cells for all canonical HIF - 2 binding sites in either cell type . No significant increase ( Wilcoxon rank sum test ) in binding of either isoform was observed following deletion of the

B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

HIF-2α ChIP-seq signal in the HIF-1α KO cells was plotted against that in the wild-type cells for all canonical HIF-2 binding sites in either cell type. No significant increase (Wilcoxon rank sum test) in binding of either isoform was observed following deletion of the other


Box - and - whisker plots showing : ( D ) The effect of HIF - 2α inactivation on HIF - 1α ChIP - seq signal at sites that specifically bound HIF - 2α , but not HIF - 1α in wild - type cells showing a slight , but significant ( p = 4 x 10 - 5 , Wilcoxon signed rank test ) decrease rather than increase ( compare red boxes ) consistent with the reduction in HIF - 1α protein observed in panel

O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

Box-and-whisker plots showing: (D) The effect of HIF-2α inactivation on HIF-1α ChIP-seq signal at sites that specifically bound HIF-2α, but not HIF-1α in wild-type cells showing a slight, but significant (p = 4 x 10-5, Wilcoxon signed rank test) decrease rather than increase (compare red boxes) consistent with the reduction in HIF-1α protein observed in panel A


The effect of HIF - 1α inactivation on HIF - 2α ChIP - seq signal ( compare blue boxes ) at sites that specifically bound HIF - 1α , but not HIF - 2α in wild - type cells showing no significant effect ( p = 0 . 5 , Wilcoxon signed rank test )

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O

The effect of HIF-1α inactivation on HIF-2α ChIP-seq signal (compare blue boxes) at sites that specifically bound HIF-1α, but not HIF-2α in wild-type cells showing no significant effect (p = 0.5, Wilcoxon signed rank test)


The effect , at sites that bind both isoforms in wild - type cells , of ( F ) HIF - 2α inactivation on HIF - 1α binding intensity showing a similar small but significant reduction rather than increase ( p = 2 x 10 - 16 , Wilcoxon Signed rank test ) as

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O

The effect, at sites that bind both isoforms in wild-type cells, of (F) HIF-2α inactivation on HIF-1α binding intensity showing a similar small but significant reduction rather than increase (p = 2 x 10-16, Wilcoxon Signed rank test) as abov


The effect , at sites that bind both isoforms in wild - type cells , o HIF - 1α inactivation on HIF - 2α binding intensity showing no significant effect ( p = 0 . 4 , Wilcoxon signed rank test )

O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

The effect, at sites that bind both isoforms in wild-type cells, o HIF-1α inactivation on HIF-2α binding intensity showing no significant effect (p = 0.4, Wilcoxon signed rank test)


Canonical HIF - 1 and HIF - 2 binding sites were determined by overlap of HIF - α and HIF - 1β binding sites identified by the MACS peak caller as for Figure 4 . The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for ( A ) HIF - 1 and ( B ) HIF - 2 sites in HKC - 8 cells , ( C ) HIF - 1 and ( D ) HIF - 2 sites in RCC4 cells , ( E ) HIF - 1 and ( F ) HIF - 2 sites in HepG2 cells . The distribution of HIF - 1 binding sites was consistently more promoter proximal than HIF - 2 sites despite the sites themselves differing between cell

O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

Canonical HIF-1 and HIF-2 binding sites were determined by overlap of HIF-α and HIF-1β binding sites identified by the MACS peak caller as for Figure 4. The distance from each binding site to the nearest annotated gene promoter was determined and plotted as a frequency distribution for (A) HIF-1 and (B) HIF-2 sites in HKC-8 cells, (C) HIF-1 and (D) HIF-2 sites in RCC4 cells, (E) HIF-1 and (F) HIF-2 sites in HepG2 cells. The distribution of HIF-1 binding sites was consistently more promoter proximal than HIF-2 sites despite the sites themselves differing between cell types


The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF - 1 ( p ~ 10 - 50 ) and HIF - 2 ( p ~ 10 - 80 ) binding sites ( Chi - squared test )

O O O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O

The distribution of accessible HRE motifs was also plotted in HepG2 cells and differed significantly from that of both HIF-1 (p~10-50) and HIF-2 (p~10-80) binding sites (Chi-squared test)


Immunoblots showing HIF - 1α , HIF - 2α and HIF - 1β protein levels in HKC - 8 , HepG2 , RCC4 and MCF - 7 cell lines in normoxia and following 16 hours incubation at 0 . 5 %

B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O

Immunoblots showing HIF-1α, HIF-2α and HIF-1β protein levels in HKC-8, HepG2, RCC4 and MCF-7 cell lines in normoxia and following 16 hours incubation at 0.5% oxygen


Sites that bound ( B ) canonical HIF - 1 ( i . e . identified in both HIF - 1α and both HIF - 1β ChIP - seq experiments ) or ( C ) canonical HIF - 2 ( i . e . identified in both HIF - 2α and both HIF - 1β ChIP - seq experiments ) were identified from ChIP - seq datasets in HepG2 and HKC - 8 cells following 16 hours incubation in 0 . 5 % oxygen and from normoxic RCC4 cells and compared with previously published data from MCF - 7 cells . ( D ) The proportion of total HIF - 1 or HIF - 2 sites that were shared between 2 , 3 or 4 cell lines is plotted . For sites that are shared between ( E ) HepG2 and HKC - 8 cells , ( F ) HKC - 8 and RCC4 cells , or ( G ) RCC4 and HepG2 cells , the ratio of HIF - 1α / HIF - 2α signal for one cell line was plotted against the other . The ratio of HIF - 1α / HIF - 2α in one cell line correlates well with that in the other . ChIP - seq signal is averaged across two independent

O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

Sites that bound (B) canonical HIF-1 (i.e. identified in both HIF-1α and both HIF-1β ChIP-seq experiments) or (C) canonical HIF-2 (i.e. identified in both HIF-2α and both HIF-1β ChIP-seq experiments) were identified from ChIP-seq datasets in HepG2 and HKC-8 cells following 16 hours incubation in 0.5% oxygen and from normoxic RCC4 cells and compared with previously published data from MCF-7 cells. (D) The proportion of total HIF-1 or HIF-2 sites that were shared between 2, 3 or 4 cell lines is plotted. For sites that are shared between (E) HepG2 and HKC-8 cells, (F) HKC-8 and RCC4 cells, or (G) RCC4 and HepG2 cells, the ratio of HIF-1α/HIF-2α signal for one cell line was plotted against the other. The ratio of HIF-1α/HIF-2α in one cell line correlates well with that in the other. ChIP-seq signal is averaged across two independent experiments


Heatmaps showing average ( n = 2 , independent ChIP - seq experiments ) ChIP - seq signal ( read counts per million mapped reads , CPM ; expressed as colour intensity ) at canonical HIF - 1 and HIF - 2 binding sites and across the flanking ± 5kb regions ( x - axis ) in HepG2 cells incubated in 0 . 5 % oxygen for 16 hours . Sites are ordered on the y - axis according to HIF signal derived from HepG2 cells incubated in hypoxia . ChIP - seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE O O O

Heatmaps showing average (n = 2, independent ChIP-seq experiments) ChIP-seq signal (read counts per million mapped reads, CPM; expressed as colour intensity) at canonical HIF-1 and HIF-2 binding sites and across the flanking ±5kb regions (x-axis) in HepG2 cells incubated in 0.5% oxygen for 16 hours. Sites are ordered on the y-axis according to HIF signal derived from HepG2 cells incubated in hypoxia. ChIP-seq signal for histone modifications was obtained from ENCODE and was derived from HepG2 cells incubated in normoxia


Bar chart showing enrichment of transcription factor binding sites ( TFBS ) at canonical HIF - 1 and HIF - 2 sites in HepG2 cells ( identified as in Figure 4 ) . TFBSs for 61 additional DNA - binding proteins were determined from ENCODE ChIP - seq data in normoxic HepG2 cells . The significance of the overlap between each set of binding sites and canonical HIF - 1 and HIF - 2 sites , was determined using a hypergeometric test . Accessible sites determined from ENCODE DNAse - seq data in HepG2 cells was used as a negative

O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O

Bar chart showing enrichment of transcription factor binding sites (TFBS) at canonical HIF-1 and HIF-2 sites in HepG2 cells (identified as in Figure 4). TFBSs for 61 additional DNA-binding proteins were determined from ENCODE ChIP-seq data in normoxic HepG2 cells. The significance of the overlap between each set of binding sites and canonical HIF-1 and HIF-2 sites, was determined using a hypergeometric test. Accessible sites determined from ENCODE DNAse-seq data in HepG2 cells was used as a negative control


Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive patient ; right : negative patient ; representative data are shown . The gating strategy is depicted in Supplementary Fig S1 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.


Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype .

O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O

Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.


Median fluorescence intensity of surface CXCR4 expression relative to isotype control ( n = 7 - 10 patients ) . Horizontal bars indicate mean of all individual patient values ; asterisks indicate statistical significance ( plasma cells vs B cells : P = 0 . 0082 ; plasma cells vs T cells : P = 0 . 0091 ; plasma cells vs CD34 + cells : P = 0 . 011 ; plasma cells vs monocytes : P = 0 . 0423 ; Student ' s t - test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype ) . MM patients judged positive for CXCR4 expression were selected for this analysis .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.


Representative MM bone marrow staining of a patient positive for MM CXCR4 expression : hematoxylin and eosin ( H & amp ; amp ; E ) staining . Immunohistochemistry for CD138 , CXCR4 , light chain kappa , and light chain lambda .

O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O

Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Real - time PCR analysis of cxcr4 transcript expression levels in HeLa ( negative control ) and in MM cell lines MM . 1S and OPM - 2 . Shown is the mean relative expression ± SEM ( n = 3 independent experiments ) . Values are normalized to the expression of ubiquitin ( Ub ) . The asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; Student ' s t - test ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O

Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).


CXCR4 protein expression assessed by immunoblotting ( one representative blot out of 3 is shown ) .

B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O

CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).


Binding of [ 68Ga ] Pentixafor to MM . 1S and OPM - 2 cells after the indicated incubation periods ( n = 3 per cell line and time point ) . Shown is the mean ± SEM . The difference between the OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O

Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).


Mean tumor - to - background ratio ( TBR ) for [ 18F ] FDG ( left ) and for [ 68Ga ] Pentixafor ( right ) in MM . 1S and OPM - 2 xenograft - bearing NOD SCID mice . Shown is the mean ± SEM , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG and * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - test ) . One - way ANOVA revealed significant differences between the groups ; P < 0 . 0001 ( not graphically shown ) .

O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).


Representative [ 68Ga ] Pentixafor PET images of three mice bearing MM . 1S ( right shoulder ) and OPM - 2 ( left shoulder ) tumors .

Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.


Flow cytometric quantification of CXCR4 cell surface expression on resected MM . 1S and OPM - 2 tumors . Data are the mean ± SEM , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - test .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.


Correlation of [ 68Ga ] Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry . n = 8 tumors were analyzed .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O

Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.


Mice ( n = 4 ) bearing OPM - 2 and MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . The white arrows point to the bladder . Quantification is shown in Supplementary Fig S3A . Source data are available online for this figure .

B-ORGANISM O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-ORGANISM O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O

Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


[ 68Ga ] PentixaforPET / CT and [ 18F ] FDGPET / CTA - D Maximum intensity projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) and [ 18F ] FDGPET / CT ( B ) of a 68 - year - old male with histologically proven multiple myeloma indicating the better lesion - to - background contrast for [ 68Ga ] Pentixafor in the corresponding myeloma manifestations . Trans - axial views of the upper thorax ( C ) and the pelvis ( D ) underline the higher uptake values of the bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Visual comparison of [ 18F ] FDG - and [ 68Ga ] Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer ( total : n = 14 ) . Number of patients ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , and with dual imaging providing complementary visual information ( complementary , n = 2 ) .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


[ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) and CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . The increased [ 68Ga ] Pentixaforbone correlates with the hyperintense T2 STIR signal ; however , the myeloma manifestations are underestimated in the corresponding bone window CT .

O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O

[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


( A ′ ) HeLa cells transfected with pEGFP - RAB11 were fixed in basal conditions and stained for mATG9 . Pearson ' s coefficient : 0 . 452 ± 0 . 034 ( SEM ) . ( A ″ ) HeLa cells were transfected with ATG9L1 - pEGFP , fixed in basal condition , and labeled for RAB11 . Pearson ' s coefficient : 0 . 413 ± 0 . 020 ( SEM ) .

O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(A′) HeLa cells transfected with pEGFP-RAB11 were fixed in basal conditions and stained for mATG9. Pearson's coefficient: 0.452 ± 0.034 (SEM).(A″) HeLa cells were transfected with ATG9L1-pEGFP, fixed in basal condition, and labeled for RAB11. Pearson's coefficient: 0.413 ± 0.020 (SEM).


( B ) HeLa cells were incubated for 1 hr at 37 ° C or 18 ° C to reach the correct temperature , loaded with transferrin Alexa - 555 for 1 hr , fixed , and labeled with anti - ATG9 and anti - TGN46 ( marker for Trans Golgi Network ) . Histogram shows Pearson ' s coefficient between ATG9 and transferrin ( Tf ) or TGN46 . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show merge at higher magnification .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(B) HeLa cells were incubated for 1 hr at 37°C or 18°C to reach the correct temperature, loaded with transferrin Alexa-555 for 1 hr, fixed, and labeled with anti-ATG9 and anti-TGN46 (marker for Trans Golgi Network). Histogram shows Pearson's coefficient between ATG9 and transferrin (Tf) or TGN46. Error bar, SEM. ∗∗∗p < 0.001. Inserts show merge at higher magnification.


( C ) HeLa cells were incubated in serum - free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa - 555 . Cells were then fixed and labeled for mATG9 and TGN46 . Pearson ' s coefficient between mATG9 and transferrin or TGN46 was quantified . Error bar , SEM . ∗ ∗ ∗ p < 0 . 001 . Inserts show the merge at higher magnification .

O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HeLa cells were incubated in serum-free medium containing Dynasore 100 μM or DMSO for 2 hr and loaded for 30 min with transferrin Alexa-555. Cells were then fixed and labeled for mATG9 and TGN46. Pearson's coefficient between mATG9 and transferrin or TGN46 was quantified. Error bar, SEM. ∗∗∗p < 0.001. Inserts show the merge at higher magnification.


( D ) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy . Pictures are at the same magnification and brightness . Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane .

O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O

(D) Cells treated or not treated with Dynasore or transfected with Dynamin K44A mutant or AP2 siRNA were fixed and then analyzed by TIRF microscopy. Pictures are at the same magnification and brightness. Cells transfected with Dynamin mutant were identified by abnormal transferrin accumulation at the plasma membrane.


( E ) HeLa cells were incubated for 1 hr at 4 ° C with ice - cold NHS - LC - Biotin ( Pierce ) solution , rinsed , and lysed in RIPA buffer . Biotinylated proteins were precipitated using streptavidin - agarose beads ( Pierce ) and blotted for mATG9 to assess protein localization at the plasma membrane .

O O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

(E) HeLa cells were incubated for 1 hr at 4°C with ice-cold NHS-LC-Biotin (Pierce) solution, rinsed, and lysed in RIPA buffer. Biotinylated proteins were precipitated using streptavidin-agarose beads (Pierce) and blotted for mATG9 to assess protein localization at the plasma membrane.


( F ) HeLa cells treated as in ( C ) were processed for immunogold labeling on cryosections and stained with anti - mATG9 antibody . Arrows show clathrin - coated structures . Scale bar in a and c , 150 nm ; scale bar in b , 100 nm .

O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(F) HeLa cells treated as in (C) were processed for immunogold labeling on cryosections and stained with anti-mATG9 antibody. Arrows show clathrin-coated structures. Scale bar in a and c, 150 nm; scale bar in b, 100 nm.


( G ) HeLa cells transfected with GFP - ATG16L1 were fixed , labeled for mATG9 , and then analyzed by TIRF microscopy . Control cells were cotransfected with GFP - ATG16L1 and Straw - ATG16L1 . Pictures are at the same magnification and brightness . See Figure S1J for quantification .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(G) HeLa cells transfected with GFP-ATG16L1 were fixed, labeled for mATG9, and then analyzed by TIRF microscopy. Control cells were cotransfected with GFP-ATG16L1 and Straw-ATG16L1. Pictures are at the same magnification and brightness. See Figure S1J for quantification.