Figure 3SETD1A FLOS domain - binding proteins were identified by Co - IP MS . Full - length SETD1A and FO3 fragment were used as baits and overlapped proteins ( red dots ) were identified as SETD1A FLOS domain - binding proteins . 293T cells were transfected with full - length SETD1A alanine mutants and protein extracts were used for the Co - IP experiments . F . MOLM - 13 leukemia cells were examined by ChIP - seq analyses against HA - tagged SETD1A , BuGZ , BUB3 , H3K4me3 and H3K4me1 . Heatmaps of SETD1A ( + ) / BuGZ ( + ) regions ( upper ) and SETD1A ( - ) / BuGZ ( + ) regions ( lower ) are shown .

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Figure 3SETD1A FLOS domain-binding proteins were identified by Co-IP MS. Full-length SETD1A and FO3 fragment were used as baits and overlapped proteins (red dots) were identified as SETD1A FLOS domain-binding proteins.293T cells were transfected with full-length SETD1A alanine mutants and protein extracts were used for the Co-IP experiments.F. MOLM-13 leukemia cells were examined by ChIP-seq analyses against HA-tagged SETD1A, BuGZ, BUB3, H3K4me3 and H3K4me1. Heatmaps of SETD1A(+)/BuGZ(+) regions (upper) and SETD1A(-)/BuGZ(+) regions (lower) are shown.


Figure 4Relative expression levels of Bugz were analyzed in Bugz shRNA - expressing mouse MLL - AF9 leukemia cells at day 3 post - treatment , respectively . Data from 3 biological replicates are shown . Data information : data are presented as mean ± SD . * * P ≤ 0 . 01 ( Student ' s t - testSurvival of recipient mice harboring dox - inducible shRNA - expressing leukemia cells was plotted ( n = 8 - 10 / group ) . Data information : * * P ≤ 0 . 01 Kaplan - Meier for ( H ) ) . Images are shown of representative karyotypes in control , Bugz and Bub3 shRNA - expressing leukemia cells . Asterisks in images indicate the trisomy 15 in all cells analyzed . Red arrows indicate chromosomal aneuploidies . Representative images of sister - chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels .

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Figure 4Relative expression levels of Bugz were analyzed in Bugz shRNA-expressing mouse MLL-AF9 leukemia cells at day 3 post-treatment, respectively. Data from 3 biological replicates are shown. Data information: data are presented as mean ± SD. **P ≤ 0.01 (Student's t-testSurvival of recipient mice harboring dox-inducible shRNA-expressing leukemia cells was plotted (n = 8-10 / group). Data information: **P ≤ 0.01 Kaplan-Meier for (H)).Images are shown of representative karyotypes in control, Bugz and Bub3 shRNA-expressing leukemia cells. Asterisks in images indicate the trisomy 15 in all cells analyzed. Red arrows indicate chromosomal aneuploidies. Representative images of sister-chromatid separation or aneuploidy in Bub3 shRNAs are shown in lower panels.


Figure 5293T cells were transfected with SETD1A F2 alanine mutant constructs . Myc - DDK - tagged SETD1A was used for Co - IP . 293T cells were transfected with BuGZ mutants . Myc - DDK - tagged BuGZ mutants illustrated on Figure S5B were used for Co - IP . 293T cells were transfected with BUB3 mutants and protein extracts were used for the Co - IP experiments . Myc - DDK - tagged BUB3 mutants illustrated on Figure S5C were used . 293T cells were transfected with Myc - DDK - tagged human BuGZ constructs and the protein extracts were used for a Co - IP experiment .

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Figure 5293T cells were transfected with SETD1A F2 alanine mutant constructs. Myc-DDK-tagged SETD1A was used for Co-IP.293T cells were transfected with BuGZ mutants. Myc-DDK-tagged BuGZ mutants illustrated on Figure S5B were used for Co-IP.293T cells were transfected with BUB3 mutants and protein extracts were used for the Co-IP experiments. Myc-DDK-tagged BUB3 mutants illustrated on Figure S5C were used.293T cells were transfected with Myc-DDK-tagged human BuGZ constructs and the protein extracts were used for a Co-IP experiment.


Figure 6293T cells were transfected with Myc - DDK - tagged BuGZ variant constructs and the protein extracts were used for the Co - IP experiment . 293T cells were transfected with Myc - DDK - tagged BuGZ constructs and the protein extracts were used for a Co - IP experiment . 293T cells were transfected with Myc - DDK - tagged wild - type BuGZ and the cells were treated with 1 , 6 - hexanediol for 3 h . The protein extracts were used for the Co - IP experiments .

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Figure 6293T cells were transfected with Myc-DDK-tagged BuGZ variant constructs and the protein extracts were used for the Co-IP experiment.293T cells were transfected with Myc-DDK-tagged BuGZ constructs and the protein extracts were used for a Co-IP experiment.293T cells were transfected with Myc-DDK-tagged wild-type BuGZ and the cells were treated with 1,6-hexanediol for 3 h. The protein extracts were used for the Co-IP experiments.


Figure 7SETD1A - GFP fusion - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , then assayed for colony - forming potential . The FUCCI reporter ( mCherry - Cdt1 ( 30 - 120 ) / Citrine - Geminin ( 1 - 110 ) ) - expressing cells were used as control . Data from 3 biological replicates are shown . Data are presented as mean ± SD . SGC - expressing Setd1afl / fl ; CreER MLL - AF9 leukemia cells were treated with tamoxifen , and RNA - seq analysis was performed with sorted cells . Data are shown as log2 fold change over tamoxifen non - treated GFP - expressing control .

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Figure 7SETD1A-GFP fusion-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, then assayed for colony-forming potential. The FUCCI reporter (mCherry-Cdt1(30-120)/Citrine-Geminin(1-110))-expressing cells were used as control. Data from 3 biological replicates are shown. Data are presented as mean ± SD.SGC-expressing Setd1afl/fl;CreER MLL-AF9 leukemia cells were treated with tamoxifen, and RNA-seq analysis was performed with sorted cells. Data are shown as log2 fold change over tamoxifen non-treated GFP-expressing control.


Figure 1 ( A , B ) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice . Brain sections were prepared 1 day after BrdU injection . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ . ( C , D ) Differentiating cells are identified by staining with BrdU and DCX . Brain sections were prepared 10 days after BrdU injection . ( E , F ) Maturation of progenitor cells are assessed by staining with BrdU and NeuN . Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness , and a total of 24 sections were obtained from one hippocampus . Scale bars : 100 μm in the large panel , and 10 μm in the magnified images . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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Figure 1(A, B) Actively dividing cells labelled by BrdU and Ki67 are reduced in the SGZ of DSCR1 mutants compared to that of wild type mice. Brain sections were prepared 1 day after BrdU injection. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ.(C, D) Differentiating cells are identified by staining with BrdU and DCX. Brain sections were prepared 10 days after BrdU injection.(E, F) Maturation of progenitor cells are assessed by staining with BrdU and NeuN. Brain sections were prepared 21 days after BrdU injection Each hippocampal section was 40 μm in thickness, and a total of 24 sections were obtained from one hippocampus. Scale bars:100 μm in the large panel, and 10 μm in the magnified images. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.05, **P< 0.01.


Figure 2 ( A ) miR - 124 expression is altered in DSCR1 mutants . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animals for each condition , * P < 0 . 01 . ( B ) Promoter activity of miR - 124 in Neuro2 A cells with DSCR1 reduction or overexpression . Firefly luciferase reporter under the control of miR - 124 promoter is measured . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . ( C ) Bisulfite sequencing analyses of the miR - 124 promoter . Each CpG site is indicated , and the methylation status of two different regions are shown . Open and filled circles represent unmethylated and methylated CpG sites , respectively . The percentage of methylated CpGs among total number of CpGs is also shown . ( D ) Neuro2A cells containing DSCR1shRNA show increased activity of miR - 124 promoter measured . The miR - 124 promoter was inserted in front of the luciferase reporter . Site - directed mutation of two methylation sites ( 31 and 58 ) in the promoter shows the luciferase activity similar to that of DSCR1 reduction . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 . ( E ) Overexpression of DSCR1 reduced the luciferase activity , while removing all methylation sites in the miR - 124 promoter increases it . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 .

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Figure 2(A) miR-124 expression is altered in DSCR1 mutants. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animals for each condition, *P< 0.01.(B) Promoter activity of miR-124 in Neuro2 A cells with DSCR1 reduction or overexpression. Firefly luciferase reporter under the control of miR-124 promoter is measured. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01.(C) Bisulfite sequencing analyses of the miR-124 promoter. Each CpG site is indicated, and the methylation status of two different regions are shown. Open and filled circles represent unmethylated and methylated CpG sites, respectively. The percentage of methylated CpGs among total number of CpGs is also shown.(D) Neuro2A cells containing DSCR1shRNA show increased activity of miR-124 promoter measured. The miR-124 promoter was inserted in front of the luciferase reporter. Site-directed mutation of two methylation sites (31 and 58) in the promoter shows the luciferase activity similar to that of DSCR1 reduction. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.(E) Overexpression of DSCR1 reduced the luciferase activity, while removing all methylation sites in the miR-124 promoter increases it. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05.


Figure 3 ( A ) DSCR1 binds to TET1 introns . The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA . ( B ) DSCR1 missing RNA recognition motif ( △ RRM ) does not bind to the TET1 intron 8 . ( C , D ) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4 . DSCR1 reduction increases the activity of this luciferase construct , while DSCR1 overexpression decreases its activity . In contrast , altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 . * * * P < 0 . 001 . ( E ) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants . TET1 is increased in DSCR1 knockout , while it is decreased in DSCR1 transgenic mice . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 animal for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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Figure 3(A) DSCR1 binds to TET1 introns. The binding affinity of DSCR1 to the intron 8 of TET1 decreased with increased dosage of U1 snRNA and U2 snRNA.(B) DSCR1 missing RNA recognition motif (△RRM) does not bind to the TET1 intron 8.(C, D) Schematic diagram of the luciferase reporter separated by TET1 intron 8 and 9 or GAPDH intron 3 and 4. DSCR1 reduction increases the activity of this luciferase construct, while DSCR1 overexpression decreases its activity. In contrast, altering DSCR1 levels did not affect luciferase activity of the construct containing GAPDH introns. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 for each condition, *P< 0.05, **P< 0.01. ***P< 0.001.(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.


Figure 4 ( B ) 3 weeks after lentivirus injection , neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region . The white arrow indicates Sox2 and GFP double positive cells . Scale bar , 10 μm . ( C ) The number of GFP and Sox2 double positive cells is restored to that of wild type . Scale bar : 10μm . Values are shown as mean ± SEM and tested for statistical significance by paired t - test . N = 3 animal for each condition , * P < 0 . 05 .

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Figure 4(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.


Figure 5 ( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO ( B ) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2 . 5 folds compared to that of wild type . However , Ts65Dn / DSCR1 + / - mouse showed normal level of DSCR1 mRNA transcript . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 3 for each condition , * P < 0 . 05 . As expected , the levels of TET1 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the TET1 mRNA expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 . As expected , the levels of miR - 124 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores the miR - 124 expression . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 . ( E , F ) The number proliferating progenitor neurons identified by BrdU and Ki - 67 double staining is restored in the SGZ of Ts65Dn / DSCR1 + / - mouse compared to that of Ts65Dn mouse . The white box area is magnified in the lower panels : DAPI ( blue ) , Ki67 ( green ) , and BrdU ( red ) . Arrow heads indicate BrdU and Ki - 67 double positive cells in the SGZ . Scale bars : 100 μm in the large image and 10 μm in magnified image . Each hippocampal section was 40 μm in thickness , and total 6 hippocampi were used for analysis . Values are shown as mean ± SEM and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 05 . ( G - J ) Ts65Dn has learning and memory defects , but Ts65Dn / DSCR1 + / - mouse clearly rescues learning and memory defects to control levels . Values are shown as mean ± SEM , and tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc test . N = 8 ( control ) , 5 ( Ts65Dn ) , 10 ( Ts65Dn / DSCR1 + / - ) animals , * P < 0 . 001 ( G , H ) . * P < 0 . 05 ( I ) , * * P < 0 . 01 ( I ) . ( J ) Open - field analysis shows no defects in movement of tested animals . Values are shown as mean ± SEM , N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) animals .

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Figure 5(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.


Figure 1C , Microscale thermophoresis ( MST ) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711 . D , dDATmfc did not show any interactions with NO711 in the MST experiments . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m . E , SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM . F , MST experiments display no interactions of purified dDATmfc with SKF89976a . The MST profiles shown are one of two independent measurements . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m . G , Michaelis - Menten uptake kinetics of 3H - GABA by the GAT1WT used in the study displaying a KM value of 11 . 4 μM . H , Inhibition of 3H - GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14 . 4 μM . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m . I , Inhibition of 3H - GABA uptake through GAT1WT by tiagabine ( Ki = 725 nM ) , NO711 ( 1 . 07 μM ) and SKF89976a ( 7 . 3 μM ) . Data information : All measurements of inhibition potency were the result of two independent experiments performed in triplicate ( n = 6 ) and all the points were used for calculating the values where the error bars represent s . e . m .

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Figure 1C, Microscale thermophoresis (MST) with purified ts5 dDATGAT construct displaying affinity of 10 µM for NO711. D, dDATmfc did not show any interactions with NO711 in the MST experiments. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.E, SKF89976a interacts with purified ts5 dDATGAT construct with an affinity of 34 µM. F, MST experiments display no interactions of purified dDATmfc with SKF89976a. The MST profiles shown are one of two independent measurements. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.G, Michaelis-Menten uptake kinetics of 3H-GABA by the GAT1WT used in the study displaying a KM value of 11.4 μM.H, Inhibition of 3H-GABA uptake by the substrate analogue nipecotic acid displaying a Ki value of 14.4 μM. Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.I, Inhibition of 3H-GABA uptake through GAT1WT by tiagabine (Ki= 725 nM), NO711 (1.07 μM) and SKF89976a (7.3 μM). Data information: All measurements of inhibition potency were the result of two independent experiments performed in triplicate (n=6) and all the points were used for calculating the values where the error bars represent s.e.m.


Figure 4B , Substitution of L300 to other hydrophobic side chains and the consequences on transport activity . L300W has the maximal effect on the transport activity . The histogram represents mean of a total 6 measurements ( n = 6 ) done as two independent measurements . Individual datapoints are represented as black dots around the error bar that represents s . e . m . Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . A near 10 - fold loss of nipecotic acid inhibition potency was observed at a value of 134 . 3 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows ; nipecotic acid , Changes to Michaelis - Menten kinetics for L300F mutant that displays weakened 3H - GABA uptake activity with KM = 20 . 9 μM . The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake . Data information : All data plots were performed as two independent experiments each time done in triplicate ( n = 6 ) . Error bars in the data display s . e . m . Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t - test display p values as follows panel E - 0 . 0001 ; tiagabine , panel F - 0 . 0017 ; NO711 , panel G - 0 . 018 ;

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B, Substitution of L300 to other hydrophobic side chains and the consequences on transport activity. L300W has the maximal effect on the transport activity. The histogram represents mean of a total 6 measurements (n=6) done as two independent measurements. Individual datapoints are represented as black dots around the error bar that represents s.e.m.Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. A near 10-fold loss of nipecotic acid inhibition potency was observed at a value of 134.3 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows; nipecotic acid,Changes to Michaelis-Menten kinetics for L300F mutant that displays weakened 3H-GABA uptake activity with KM = 20.9 μM. The inhibition potencies of the inhibitors display lowered value suggesting an improved ability to compete for the GABA uptake. Data information: All data plots were performed as two independent experiments each time done in triplicate (n=6). Error bars in the data display s.e.m. Statistical significance of inhibition potencies were measured between the mutant and GAT1WT transporter measured with an unpaired t-test display p values as follows panel E-0.0001; tiagabine, panel F-0.0017; NO711, panel G-0.018; SKF89976a


Figure 5E , F , Eadie - Hofstee plots of E , tiagabine and F , NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT . G , Eadie - Hofstee plot of SKF89976a displaying mixed ( competitive + non - competitive ) inhibition of 3H - GABA uptake in GAT1WT . Data information : ( E - G ) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors . Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates . Error bars represent s . e . m .

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Figure 5E, F, Eadie-Hofstee plots of E, tiagabine and F, NO711 display competitive mode of inhibition of 3H GABA uptake in GAT1WT. G, Eadie-Hofstee plot of SKF89976a displaying mixed (competitive+non-competitive) inhibition of 3H-GABA uptake in GAT1WT. Data information: (E-G) Concentrations of the inhibitors were decided based on the Ki values calculated for individual inhibitors. Each data point was plotted using 6 measurements carried out in two independent measurements in triplicates. Error bars represent s.e.m.


Figure 6C , Michaelis - Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value ( 28 . 54 ± 2 . 3 μM ) . The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m . D , 3H - GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30 . 1 μM Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m . E , The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT . Data information : The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate . The error bars represent s . e . m .

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Figure 6C, Michaelis-Menten kinetics of GAT1WT and GAT1 S359E displays a weakened KM value (28.54 ± 2.3 μM). The enhanced Vmax is a consequence of the enhanced expression of the mutant transporter in the assayed cells monitored by FSEC. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.D, 3H-GABA uptake inhibition by the substrate analogue nipecotic acid for GAT1 S359E displaying a Ki value of 30.1 μM Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.E, The equivalent mutation in dDATWT E384S causes a complete ablation of transport activity despite retaining similar expression levels as dDATWT. Data information: The kinetic plots were plotted as a mean of 6 measurements carried out in two independent measurements in triplicate. The error bars represent s.e.m.


figf5a , GFP - MAP1LC3B foci after RNAi and / or rapamycin treatment in U2OS cells . Scale bar , 20 µm .

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figf5a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.


Figure 1A LC3 punctum formation in WT and GCN5 KO HeLa cells ( Scale bars , 10 µm ) . Immunoblot showing LC3 - II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf . Immunoblot showing LC3 - II formation MB - 3 - treated HeLa cells in the presence or absence of the lysosome inhibitor Baf . D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) . E Quantification of LC3 puncta in cells The cells were treated with or without Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) . Formation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf treatmentFormation of LC3 puncta in HeLa cells overexpressing GFP - GCN5 with or without Baf treatment ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bar , 10 µm ) . H LC3 - II formation in GFP - GCN5 - overexpressing HeLa cells . I GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were cultured with GCN5 siRNA with or without CQ . J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP - GCN5 and addition of CQ . K Representative images of mCherry - Atg8a ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) using the pan - fat body driver ( cg - GAL4 ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - test ; Scale bars , 10 µm ) .

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Figure 1A LC3 punctum formation in WT and GCN5 KO HeLa cells (Scale bars, 10 µm).Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.Immunoblot showing LC3-II formation MB-3-treated HeLa cells in the presence or absence of the lysosome inhibitor Baf.D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).E Quantification of LC3 puncta in cells The cells were treated with or without Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatmentFormation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bar, 10 µm).H LC3-II formation in GFP-GCN5-overexpressing HeLa cells.I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ.J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ.K Representative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, ***p < 0.001, Student's t-test; Scale bars, 10 µm).


Figure 2A LAMP1 puncta ( green ) and DAPI ( blue ) in WT and GCN5 KO HEK293 cells ( Scale bars , 10 µm ) . B Fluorescence - activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker . Fluorescence intensity of 10 , 000 cells per sample was measured . C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells . CTSD HC , cathepsin D heavy chain . D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) . E Quantification of LAMP1 puncta in ( A ) and ( D ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) . F Hexosaminidase activity in GCN5 KO HEK293 cells ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) . G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ . H Representative images of LysoTracker staining ( red ) and DAPI ( blue ) in Drosophila larval fat body in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) . Drosophila ( cg - GAL4 / + ) was used as the control ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - test ; Scale bars , 10 µm ) .

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Figure 2A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm).B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured.C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells. CTSD HC, cathepsin D heavy chain.D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).E Quantification of LAMP1 puncta in (A) and (D) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).F Hexosaminidase activity in GCN5 KO HEK293 cells (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ.H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).


Figure 3A Quantification of LC3 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) . B Immunoblot showing LC3 - II formation in WT , GCN5 KO and GCN5 / TFEB DKO HeLa cells in the presence or absence of Baf . C Quantification of LAMP1 puncta in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) . D Immunoblot showing LAMP1 levels in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells . E Acetylation of TFEB - Flag in stable TFEB - Flag - expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated . TFEB - Flag was immunoprecipitated with anti - Flag beads and analyzed by immunoblot using anti - acetyl - lysine ( Ace - lys ) . F TFEB - Flag acetylation in the stable TFEB - Flag - expressing HEK293 cells transfected with Myc - tagged GCN5 or GCN5 - E575Q after incubation with GCN5 siRNA for 48 h . G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB - 3 or the p300 / CBP inhibitor C646 . H3 , histone H3 . Ace - H3 ( K9 ) and Ace - H3 ( K27 ) were used to show the activity of GCN5 and p300 , respectively . H Co - precipitation of GCN5 with TFEB . TFEB was immunoprecipitated from MEF cells using anti - TFEB , and the precipitates were analyzed using anti - GCN5 . I In vitro acetylation assays of TFEB . Purified recombinant TFEB was incubated with Myc - GCN5 or Myc - GCN5 - E575Q immunoprecipitated from HEK293 cells . J Acetylation of Flag - tagged TFEB or TFEB mutants expressed in HEK293 cells . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg .

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Figure 3A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf.C Quantification of LAMP1 puncta in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).D Immunoblot showing LAMP1 levels in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells.E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys).F TFEB-Flag acetylation in the stable TFEB-Flag-expressing HEK293 cells transfected with Myc-tagged GCN5 or GCN5-E575Q after incubation with GCN5 siRNA for 48 h.G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively.H Co-precipitation of GCN5 with TFEB. TFEB was immunoprecipitated from MEF cells using anti-TFEB, and the precipitates were analyzed using anti-GCN5.I In vitro acetylation assays of TFEB. Purified recombinant TFEB was incubated with Myc-GCN5 or Myc-GCN5-E575Q immunoprecipitated from HEK293 cells.J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.


Figure 4A RT - qPCR analysis of the expression of TFEB target genes in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test . B RT - qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5 . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test . C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB - WT or TFEB - 3KR . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test . D , E Luciferase activity measured in WT , GCN5 KO and GCN5 / TFEB DKO HEK293 cells ( D ) , and in HEK293 cells with TFEB - WT or TFEB - 3KR transfection ( E ) . The cells were transfected or co - transfected with a TFEB - luciferase reporter . 3KR : Lys 116 , Lys 274 , and Lys 279 were replaced by Arg . Data information : In this Figure , data are presented as mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

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Figure 4A RT-qPCR analysis of the expression of TFEB target genes in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.B RT-qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB-WT or TFEB-3KR. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.D, E Luciferase activity measured in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (D), and in HEK293 cells with TFEB-WT or TFEB-3KR transfection (E). The cells were transfected or co-transfected with a TFEB-luciferase reporter. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


Figure 5A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 ( Scale bars , 10 µm ) . B Subcellular localization of Flag - tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1 . Cells were stained with anti - Flag antibody ( Scale bars , 10 µm ) . C ChIP - qPCR analysis of TFEB binding to the promoter of its target genes CLCN7 , GLA and CTSD . Normal mouse IgG and primers against the upstream region lacking CLEAR sites ( up ) were used as negative controls ( mean ± SEM ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test ) . D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA . A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants , and was subjected to electrophoresis . Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non - binding DNA control . F Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - TFEB in HEK293T cells . Immunoprecipitation was carried out with anti - Myc beads , and the precipitates were analyzed using anti - Flag . Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged MITF ( G ) in HEK293T cells . Co - precipitation of Flag - tagged TFEB and TFEB mutants with Myc - tagged TFE3 ( H ) in HEK293T cells . I TFEB homodimer formation detected by glutaraldehyde ( GA ) crosslinking . Purified recombinant GST - tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde , then the products were analyzed by immunoblotting using anti - TFEB .

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Figure 5A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm).B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm).C ChIP-qPCR analysis of TFEB binding to the promoter of its target genes CLCN7, GLA and CTSD. Normal mouse IgG and primers against the upstream region lacking CLEAR sites (up) were used as negative controls (mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test).D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control.F Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-TFEB in HEK293T cells. Immunoprecipitation was carried out with anti-Myc beads, and the precipitates were analyzed using anti-Flag.Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged MITF (G) in HEK293T cells.Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged TFE3 (H) in HEK293T cells.I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.


Figure 6A Acetylation of TFEB - Flag , Myc - GCN5 and histone H3 in HEK293 cells . The cells were either starved of glucose or amino acids , or treated with Torin1 or MB - 3 . B Acetylation of endogenous TFEB in HeLa cells . C GCN5 activity measured by in vitro acetylation assay using Myc - GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate . ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) . D Co - precipitation of TFEB - Flag with Myc - TFEB in HEK293 cellsE LC3 - II formation in HeLa cells transfected with Flag - tagged TFEB or TFEB mutants after TFEB RNAi . The cells were treated with or without amino acid starvation . F GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were treated with or without addition of CQ . Representative images of mCherry - Atg8a ( red ) ( G ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) . Representative images of LysoTracker ( red ) staining ( H ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) . Quantification of mCherry - ATG8a puncta ( I ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) . Quantification of LysoTracker puncta ( J ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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Figure 6A Acetylation of TFEB-Flag, Myc-GCN5 and histone H3 in HEK293 cells. The cells were either starved of glucose or amino acids, or treated with Torin1 or MB-3.B Acetylation of endogenous TFEB in HeLa cells.C GCN5 activity measured by in vitro acetylation assay using Myc-GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate. (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).D Co-precipitation of TFEB-Flag with Myc-TFEB in HEK293 cellsE LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).Quantification of mCherry-ATG8a puncta (I) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Figure 7The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding . The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as the control . The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding . The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as the control . C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR - Gal4 driver . The Drosophila were fed with or without CQ . Drosophila ( GMR - Gal4 / + ) was used as the control . D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ ( 30 flies per condition were randomly selected for analyses ; * p < 0 . 05 , * * * p < 0 . 001 , ꭓ2 test ) .

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Figure 7The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).


Figure 1D . Quantity of Ki67 + cells in tumour and representative histology images analysed by Immunohistochemistry ( IHC ) that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates . F . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 , Muc1 of colon tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates . G . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein expression ( right ) in the tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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Figure 1D. Quantity of Ki67+ cells in tumour and representative histology images analysed by Immunohistochemistry (IHC) that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10, Muc1 of colon tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.G. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein expression (right) in the tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


Figure 2representative histological images ( B ) of wild - type control mice or BTNL2 - KO mice treated with 2 % DSS for 8 days were shown ( n = 15 ) . E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β , IL - 6 and IL - 10 of colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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Figure 2representative histological images (B) of wild-type control mice or BTNL2-KO mice treated with 2% DSS for 8 days were shown (n=15).E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β, IL-6 and IL-10 of colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


Figure 3representative H & E images ( H ) of wild - type control mice or BTNL2 - KO mice treated with Fc or mIL - 22 - Fc ( ip . 5 μg / mouse ) at day 0 , 2 , 4 , 6 during 2 % DSS treatment were shown ( n = 12 ) . Data information Data are representative of three biological replicates H ) .

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Figure 3representative H&E images (H) of wild-type control mice or BTNL2-KO mice treated with Fc or mIL-22-Fc (ip. 5 μg/mouse) at day 0, 2, 4, 6 during 2% DSS treatment were shown (n=12). Data information Data are representative of three biological replicates H).


Figure 4B . IL - 22 and IL - 17a expression in lamina propria lymphocytes ( LPLs ) after cultured in plate - coated Fc , mBTNL2 - Fc ( 30 μg / mL ) with or without IL - 23 ( 10 ng / mL ) for 20 h was analyzed by ELISA . C . IL - 22 production from cells of LPLs cultured in the presence of plate - coated Fc , mBTNL2 - Fc with anti - control Ab or anti - BTNL2 mAb for 20 h was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were shownD . CD4 + T cells , γδ T cells , ILC3s , CD8 + T cells sorted by FACS were cultured in the plate - coated Fc , mBTNL2 - Fc with or without IL - 23 ( 10 ng / mL ) for 20 h , and IL - 22 production was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were shownF . IL - 22 production was analyzed by Flow cytometry in LPLs of wild - type mice treated with Fc or mBTNL2 - Fc ( ip . 50 μg / mouse ) on day 0 , 2 , 4 , 6 during 2 % DSS treatment . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 12 for the Fc group and n = 14 for the mBTL2 - Fc group ( F ) . Pooled data from three biological replicates were shown F . G . IL - 22 , IL - 17a , RORC and HIF - 1α expression in splenocytes after cultured in plate - coated Fc or mBTNL2 - Fc ( 30 μg / mL ) for 20 h was analyzed by real - time PCR . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Data are representative of three biological

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. IL-22 and IL-17a expression in lamina propria lymphocytes (LPLs) after cultured in plate-coated Fc, mBTNL2-Fc (30 μg/mL) with or without IL-23 (10 ng/mL) for 20 h was analyzed by ELISA. C. IL-22 production from cells of LPLs cultured in the presence of plate-coated Fc, mBTNL2-Fc with anti-control Ab or anti-BTNL2 mAb for 20 h was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shownD. CD4+ T cells, γδ T cells, ILC3s, CD8+ T cells sorted by FACS were cultured in the plate-coated Fc, mBTNL2-Fc with or without IL-23 (10 ng/mL) for 20 h, and IL-22 production was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shownF. IL-22 production was analyzed by Flow cytometry in LPLs of wild-type mice treated with Fc or mBTNL2-Fc (ip. 50 μg/mouse) on day 0, 2, 4, 6 during 2%DSS treatment. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=12 for the Fc group and n=14 for the mBTL2-Fc group (F). Pooled data from three biological replicates were shown F.G. IL-22, IL-17a, RORC and HIF-1α expression in splenocytes after cultured in plate-coated Fc or mBTNL2-Fc (30 μg/mL) for 20 h was analyzed by real-time PCR. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Data are representative of three biological replicates


Figure 5E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 from colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 15 Pooled data from three biological replicatesJ . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 of colonic tissue described in ( F ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 12 Pooled data from three biological

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 from colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=15 Pooled data from three biological replicatesJ. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 of colonic tissue described in (F) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=12 Pooled data from three biological replicates


Figure 6Representative colonic tumour burden ( B ) that described in ( A ) were shown ( n = 12 ) . Data information : Data are representative of three biological replicates ( BF . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 and Muc1 of colonic tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for F , Each dot represents one mouse , n = 12 . Pooled data from three biological replicatesG . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein ( right ) expression of tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for G , Each dot represents one mouse , n = 12 . Data are representative of three biological replicates left panel of G ) . Pooled data from three biological replicates are shown in right panel of GH , Quantity of Ki67 + cells in tumours and representative histology images that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for H ) . Each dot represents one mouse , n = 12 . Pooled data from three biological replicates are shown in H .

O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative colonic tumour burden (B) that described in (A) were shown (n=12). Data information: Data are representative of three biological replicates (BF. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10 and Muc1 of colonic tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for F, Each dot represents one mouse, n=12. Pooled data from three biological replicatesG. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein (right) expression of tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for G, Each dot represents one mouse, n=12. Data are representative of three biological replicates left panel of G). Pooled data from three biological replicates are shown in right panel of GH, Quantity of Ki67+ cells in tumours and representative histology images that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for H). Each dot represents one mouse, n=12. Pooled data from three biological replicates are shown in H.


Figure 7A . Survival of patients with colorectal cancer from Kaplan - Meier database was analyzed Left panel of A were representative of 150 technical replicates , and right panel of A were representative of 499 technical replicates . representative histology images in human colorectal cancer specimens ( 1 # - 5 # ) and human colon polyp specimens ( 6 # - 7 # ) were shown . Number of cells expressed BTNL2 or IL - 22 was analysed in high power field ( HPF ) , arrows indicate cells expressed BTNL2 or IL - 22 ( B ) . D - E . Expression of BTNL2 in normal colon tissue or DSS + AOM induced tumour tissue were measured by immunoblot ( D ) and real - time PCR ( E ) ( n = 5 ) . Data information : All data are mean ± s . e . m . NS , not significant . , * * P < 0 . 01 based on two - tailed Student ' s t test for ( E ) . Each dot represents one mouse , n = 5 .

O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Survival of patients with colorectal cancer from Kaplan-Meier database was analyzed Left panel of A were representative of 150 technical replicates, and right panel of A were representative of 499 technical replicates.representative histology images in human colorectal cancer specimens (1#-5#) and human colon polyp specimens (6#-7#) were shown. Number of cells expressed BTNL2 or IL-22 was analysed in high power field (HPF), arrows indicate cells expressed BTNL2 or IL-22 (B).D-E. Expression of BTNL2 in normal colon tissue or DSS+AOM induced tumour tissue were measured by immunoblot (D) and real-time PCR (E) (n=5). Data information: All data are mean ± s.e.m. NS, not significant., **P < 0.01 based on two-tailed Student's t test for (E). Each dot represents one mouse, n=5.


Figure 1 ( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin . ( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) . ( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . ( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


Figure 2 ( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag . ( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control . ( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins . ( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.(B) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


Figure 3 ( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.