B NRPE1 ( red ) was visualized by its specific antibody in cells expressing FLAG ‐ tagged AGO6 ( green ) . The yellow signals due to the overlap of red and green channels in merged images indicate protein co ‐ localization . DNA ( blue ) was stained with DAPI . AGO6 ‐ NRPE1 co ‐ localized to nucleoplasmic foci ( yellow dots , marked with white arrow ) .

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B NRPE1 (red) was visualized by its specific antibody in cells expressing FLAG‐tagged AGO6 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co‐localization. DNA (blue) was stained with DAPI. AGO6‐NRPE1 co‐localized to nucleoplasmic foci (yellow dots, marked with white arrow).


A AGO6 dysfunction reduces levels of Pol V ‐ dependent transcripts . RNA levels were measured by RT - qPCR . Means ± SD are shown , n = 3 .

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A AGO6 dysfunction reduces levels of Pol V‐dependent transcripts. RNA levels were measured by RT-qPCR. Means ± SD are shown, n = 3.


B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6 . Anti ‐ NRPE1 antibody was used for ChIP assays . Means ± SD are shown , n = 3 .

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B AGO6 dysfunction decreases Pol V occupancy at chromatin of loci where Pol V transcript levels are affected by AGO6. Anti‐NRPE1 antibody was used for ChIP assays. Means ± SD are shown, n = 3.


A Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol IV . Identities of siRNAs were retrieved from Havecker et al ( ) , and siRNAs were grouped into 1 , 210 AGO4 ‐ bound clusters and 1 , 486 AGO6 ‐ bound clusters ( see Materials and Methods for details ) , each of which corresponds to a colored horizontal bar ; the bars are stacked numerically into a column ( y ‐ axis ) . Information on siRNA levels in Pol IV mutant and wild ‐ type plants was retrieved from Zhang et al ( ) .

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A Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol IV. Identities of siRNAs were retrieved from Havecker et al (), and siRNAs were grouped into 1,210 AGO4‐bound clusters and 1,486 AGO6‐bound clusters (see Materials and Methods for details), each of which corresponds to a colored horizontal bar; the bars are stacked numerically into a column (y‐axis). Information on siRNA levels in Pol IV mutant and wild‐type plants was retrieved from Zhang et al ().


B Heatmap depiction of the dependence of AGO4 ‐ and AGO6 ‐ bound siRNAs on Pol V .

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B Heatmap depiction of the dependence of AGO4‐ and AGO6‐bound siRNAs on Pol V.


C Quantification of individual 24 ‐ nt siRNAs by TaqMan small RNA assays . Double : ago4 ‐ 6 ago6 ‐ 2 double mutant . snoR101 was used as an internal control . RNA levels in the mutants were relative to those of the wild ‐ type ( Col ‐ 0 ) . Error bars indicate SD , n ≥ 3 .

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C Quantification of individual 24‐nt siRNAs by TaqMan small RNA assays. Double: ago4‐6 ago6‐2 double mutant. snoR101 was used as an internal control. RNA levels in the mutants were relative to those of the wild‐type (Col‐0). Error bars indicate SD, n ≥ 3.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were subjected to IP using anti ‐ FLAG antibodies . The resulting precipitates were examined by immunoblot analysis with anti ‐ FLAG and anti ‐ HA antibodies .

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(B,C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies.


( D ) HEK293T cells were cultured in regular or starvation medium for 2 h . Cells were harvested and treated with DSP .

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(D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP.


( E ) HEK293T cells were harvested and treated with DSP .

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(E) HEK293T cells were harvested and treated with DSP.


( F ) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h . After treatment with DSP , cell lysates were subjected to IP analysis .

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(F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis.


( G ) Atg14F / F ( undeleted ) or Atg14Δ / Δ ( deleted ) MEFs were harvested and treated with DSP . * ( E ) and * * ( G ) indicate the positions of the immunoglobulin light and heavy chains , respectively . aa , amino acid ; DSP , dithiobis ( succinimidyl propionate ) ; IP , immunoprecipitation ; MEFs , mouse embryonic fibroblasts .

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(G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.


( A ) Immunoblot analysis of Atg proteins in the indicated MEFs .

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(A) Immunoblot analysis of Atg proteins in the indicated MEFs.


( B ) m5 ‐ 7 cells were cultured in regular medium containing 10 ng / ml Dox for 8 days , and then cultured in the absence of Dox for a further 2 days .

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(B) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days.


( C , D ) m5 ‐ 7 cells were cultured in regular medium with or without 10 ng / ml Dox for 4 days , and then cultured in the presence of 50 μg / ml CHX for the indicated time periods ( C ) . mRNA levels of the indicated genes were measured by quantitative PCR following the 4 ‐ day Dox treatment . Data represent mean ± s . e . ( * P0 . 05 ) ( D ) . CHX , cycloheximide ; Dox , doxycycline ; MEFs , mouse embryonic fibroblasts ; mRNA , messenger RNA ; WT , wild ‐ type .

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(C, D) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were analysed by IP . IP , immunoprecipitation ; ND , not determined .

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(B, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were analysed by IP. IP, immunoprecipitation; ND, not determined.


( A ) WT MEFs , Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h . * indicates nonspecific immunoreactive bands .

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(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands.


( B , C ) Atg16L1 KO MEFs stably expressing GFP - ULK1 and either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM ( C ) or starvation medium ( B , C ) for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP , anti ‐ Atg16L1 and anti ‐ LC3 antibodies . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) . The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods . Data represent mean ± s . e . ( * P0 . 001 , analysis of variance followed by Bonferroni / Dunn post hoc test ) . Scale bar , 10 μm , and 2 μm in inset .

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

(B, C) Atg16L1 KO MEFs stably expressing GFP-ULK1 and either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset.


( D , E ) Cells stably expressing GFP - LC3 were cultured in regular DMEM , starvation medium or starvation medium in the presence of 0 . 2 μM WM or 100 nM BafA1 for 6 h . Total cellular GFP - LC3 signals were analysed by flow cytometry . Representative FACS data were shown ( D ) . The geometric mean of fluorescence intensity was determined . Values are expressed as a percentage of the mean of control cells cultured in regular DMEM . Data represent mean ± s . e . ( * P0 . 05 ) ( E ) . BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; KO , knockout ; MEFs , mouse embryonic fibroblasts ; NS , not significant ; WM , wortmannin ; WT , wild ‐ type .

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

(D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.


( A ) Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and subjected to immunofluorescence microscopy using anti ‐ Atg16L1 antibody . Note that full ‐ length Atg16L1 and Atg16L1 ( 1 - 230 ) , but not Atg16L1Δ ( 230 - 300 ) , showed punctate structures under starvation conditions ( inset ) .

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(A) Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1-230), but not Atg16L1Δ(230-300), showed punctate structures under starvation conditions (inset).


( B ) FIP200 KO MEFs stably expressing either CFP - Atg16L1 ( 1 - 588 ) , CFP - Atg16L1 ( 1 - 230 ) or CFP - Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP antibody . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) .

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(B) FIP200 KO MEFs stably expressing either CFP-Atg16L1(1-588), CFP-Atg16L1(1-230) or CFP-Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1-230).


( C , D ) FIP200 KO MEFs stably expressing CFP - Atg16L1 ( 1 - 230 ) were cultured in regular DMEM . Scale bar , 10 μm , and 2 μm in inset .

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(C, D) FIP200 KO MEFs stably expressing CFP-Atg16L1(1-230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset.


( a ) Representative micro - CT pictures of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Note the severe osteoporosis in A20myel - KO mice . ( b ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Each dot represents an individual mouse ( A20WT , n = 10 ; A20myel - KO , n = 8 ) .

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(a) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Note the severe osteoporosis in A20myel-KO mice. (b) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Each dot represents an individual mouse (A20WT, n=10; A20myel-KO, n=8).


( a ) Levels of TNF and IL - 6 in serum of A20WT , A20OC - KO and A20myel - KO mice . Each dot represents an individual mouse ( A20WT , n = 11 ; A20OC - KO , n = 11 and A20myel - KO , n = 14 ) . Data are expressed as mean ± SEM . *

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(a) Levels of TNF and IL-6 in serum of A20WT, A20OC-KO and A20myel-KO mice. Each dot represents an individual mouse (A20WT, n=11; A20OC-KO, n=11 and A20myel-KO, n=14). Data are expressed as mean ± SEM. *


( c ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20OC - KO littermates . Each dot represents an individual mouse ( A20WT , n = 6 ; A20OC - KO , n = 9 ) .

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(c) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Each dot represents an individual mouse (A20WT, n=6; A20OC-KO, n=9).


( e ) Representative pictures of TRAP - stained sections from tibia of 20 week - old A20WT and A20OC - KO mice . Red arrows indicate osteoclasts .

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(e) Representative pictures of TRAP-stained sections from tibia of 20 week-old A20WT and A20OC-KO mice. Red arrows indicate osteoclasts.


( f ) Quantification of the number of TRAP - positive cells on sections from tibia of 20 week - old A20WT and A20OC - KO mice . Each dot represents an individual mouse ( A20WT , n = 5 ; A20OC - KO , n = 6 ) .

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(f) Quantification of the number of TRAP-positive cells on sections from tibia of 20 week-old A20WT and A20OC-KO mice. Each dot represents an individual mouse (A20WT, n=5; A20OC-KO, n=6).


( d ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF - κB reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and with either an empty vector or a vector expressing wild - type A20 . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency .

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(d) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF-κB reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and with either an empty vector or a vector expressing wild-type A20. Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency.


( e ) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel - KO mice and stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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(e) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel-KO mice and stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( f ) Wild - type and A20 deficient BMDMs were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for RANK , A20 , TRAF6 , HOIP and Sharpin .

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(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.


( g ) BMDMs from A20WT and A20myel - KO mice were stimulated with RANKL ( 100ng / ml ) for the indicated time periods and immunoblotted to visualize p100 processing to p52 . Actin is shown as a loading control .

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(g) BMDMs from A20WT and A20myel-KO mice were stimulated with RANKL (100ng/ml) for the indicated time periods and immunoblotted to visualize p100 processing to p52. Actin is shown as a loading control.


( a ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transfected with an NF - κB luciferase reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and plasmids encoding different A20 variants : A20WT , A20DUB ( C103A mutation ) , A20ZnF4 ( C624A / C627A ) , A20ZnF7 ( C775A / C779A ) , and A20ZnF4ZnF7 ( C624A / C627A / C775A / C779A ) . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency . Data are expressed as the mean of technical triplicates ± SD .

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(a) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transfected with an NF-κB luciferase reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and plasmids encoding different A20 variants: A20WT, A20DUB (C103A mutation), A20ZnF4 (C624A/C627A), A20ZnF7 (C775A/C779A), and A20ZnF4ZnF7 (C624A/C627A/C775A/C779A). Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD.


( c - d ) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice , stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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(c-d) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice, stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( e ) BMDMs isolated from control ( A20WT ) , A20ZnF7 , A20ZnF4ZnF7 and A20DUB mice were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


( f ) Wild - type BMDMs were pretreated for 1h with HOIPIN - 8 ( 50µM ) or left untreated and stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


Immunoblots of acyl - biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation - deficient mutant form of the protein with cysteines replaced with alanine residues . The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull - down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein .

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Immunoblots of acyl-biotin exchange purifications from Hela cells transiently transfected with WT Golga7b or a palmitoylation-deficient mutant form of the protein with cysteines replaced with alanine residues. The hydroxylamine sensitivity of the Golga7b signal in the + HA condition of the WT pull-down and not in either the - HA condition or the + HA condition of the mutant shows that Golga7b is a palmitoylated protein.


Immunoblots of acyl - biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non - targeting siRNA .

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Immunoblots of acyl-biotin exchange purifications from Hela cells treated with either DHHC5 siRNA or control non-targeting siRNA.


Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant .

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Immunoblots showing rescue of Golga7b palmitoylation in DHHC5 depleted cells after transfection of WT DHHC5 but not by the DHHC5 catalytic mutant.


A significant reduction in the Golga7b signal is observed in the + HA condition of the pull - down when DHHC5 is depleted with siRNA . The graph displays mean + / - s . e . m . N = 4 , * = p < 0 . 05 , paired t - test .

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A significant reduction in the Golga7b signal is observed in the + HA condition of the pull-down when DHHC5 is depleted with siRNA. The graph displays mean +/- s.e.m. N=4, *=p<0.05, paired t-test.


Immunoblots of WT and non - palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants ( DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine , C - terminal mutant = mutant where the 3 C - terminal palmitoylation sites of DHHC5 have been mutated to alanine residues . ) . WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C - terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C - terminus of DHHC5 is essential for the stability of this interaction .

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Immunoblots of WT and non-palmitoylatable mutant Golga7b from immunoprecipitations of WT DHHC5 and a pair of DHHC5 mutants (DHHS5 = catalytically inactive mutant where catalytic cysteine has been mutated to serine, C-terminal mutant = mutant where the 3 C-terminal palmitoylation sites of DHHC5 have been mutated to alanine residues.). WT and DHHS5 immunoprecipitates both WT and mutant Golga7b but the C-terminal mutant is unable to immunoprecipitate either form of Golga7b showing that the palmitoylation of the C-terminus of DHHC5 is essential for the stability of this interaction.


Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5 . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and the catalytic mutant DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHS5 ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing WT Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Confocal images of HeLa cells co - expressing palmitoylation deficient mutant Golga7b ( 1st column ) and DHHC5 C - terminal palmitoylation deficient mutant ( 2nd column ) , with a merged image ( 3rd column ) . Quantification of the plasma membrane signal of DHHC5 in each of the images ( A - F ) , the graph displays mean + / - s . e . m . * * * * = p < 0 . 001 , n . s . = not significant , unpaired t - test . N = 35 , DHHC5 + WT Golga7b . N = 41 , DHHC5 + mutant Golga7b . N = 35 , DHHS5 + WT Golga7b . N = 31 , DHHS5 + Golga7b mutant . N = 29 , C - terminal mutant + WT Golga7b . N = 30 , C - terminal mutant + mutant Golga7b . Co expression of palmitoylation - deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane . The majority of the DHHC5 C - terminal palmitoylation - deficient mutant is localised to the plasma membrane in a Golga7b independent manner . All N numbers refer to the number of cells imaged and quantified , all experiments repeated 4 times . Data information : All images show ectopic protein expression only . All scale bars 10µm .

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Tagged constructs with palmitoylation site mutations in DHHC5 and Golga7b were expressed in in HeLa cells depleted of endogenous DHHC5. Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and the catalytic mutant DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHS5 (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing WT Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Confocal images of HeLa cells co-expressing palmitoylation deficient mutant Golga7b (1st column) and DHHC5 C-terminal palmitoylation deficient mutant (2nd column), with a merged image (3rd column). Quantification of the plasma membrane signal of DHHC5 in each of the images (A-F), the graph displays mean +/- s.e.m. ****=p<0.001, n.s. = not significant, unpaired t-test. N=35, DHHC5 + WT Golga7b. N=41, DHHC5 + mutant Golga7b. N=35, DHHS5 + WT Golga7b. N=31, DHHS5 + Golga7b mutant. N=29, C-terminal mutant + WT Golga7b. N=30, C-terminal mutant + mutant Golga7b. Co expression of palmitoylation-deficient mutant Golga7b significantly reduces DHHC5 as well as DHHCS5 signal at the plasma membrane. The majority of the DHHC5 C-terminal palmitoylation-deficient mutant is localised to the plasma membrane in a Golga7b independent manner. All N numbers refer to the number of cells imaged and quantified, all experiments repeated 4 times. Data information: All images show ectopic protein expression only. All scale bars 10µm.


Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged WT Golga7b . Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG - tagged mutant Golga7b .

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Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged WT Golga7b. Confocal images of endogenous DHHC5 in HeLa cells expressing FLAG-tagged mutant Golga7b.


Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non - targeting siRNA . Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA .

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Confocal images of endogenous Golga7b and DHHC5 in HeLa cells treated with negative control non-targeting siRNA. Confocal images of endogenous Golga7b and DHHC5 in HeLa cell treated with Golga7b siRNA.


Quantification of plasma membrane signal of endogenous DHHC5 when co - expressed with WT or mutant Golga7b or without any transfected Golga7b . * * * * = P < 0 . 001 , t - test , endogenous + WT Golga7b n = 37 , endogenous + mutant Golga7b n = 23 , endogenous n = 42 . Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell , while WT Golga7b stabilises DHHC5 at the plasma membrane .

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Quantification of plasma membrane signal of endogenous DHHC5 when co-expressed with WT or mutant Golga7b or without any transfected Golga7b. ****=P<0.001, t-test, endogenous + WT Golga7b n=37, endogenous + mutant Golga7b n=23, endogenous n=42. Expression of the palmitoylation deficient mutant Golga7b causes endogenous DHHC5 to be internalised in the cell, while WT Golga7b stabilises DHHC5 at the plasma membrane.


Quantification of the plasma membrane signal of DHHC5 in ( C and D ) , * * * * = p < 0 . 001 t - test , control siRNA n = 23 , Golga7b siRNA n = 23 .

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Quantification of the plasma membrane signal of DHHC5 in (C and D), ****=p<0.001 t-test, control siRNA n=23, Golga7b siRNA n=23.


FLAG - DHHC5 immunoprecipitations were performed using lysates from Hela cells co - expressing either wild - type ( WT ) or palmitoylation deficient mutant ( m ) Golga7b . The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b , quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 .

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FLAG-DHHC5 immunoprecipitations were performed using lysates from Hela cells co-expressing either wild-type (WT) or palmitoylation deficient mutant (m) Golga7b. The interaction of DHHC5 with its substrate Flot2 was measured by immunoblotting and is significantly reduced in the presence of palmitoylation deficient mutant Golga7b, quantification normalised to input, *=p<0.05, ratio paired t-test, n=3.


Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG - DHHC5 is expressed alone or co - expressed with either WT or mutant Golga7b . IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation .

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Venn diagram comparing the overlap of numbers of proteins immunoprecipitated when FLAG-DHHC5 is expressed alone or co-expressed with either WT or mutant Golga7b. IF images are taken from figures EV2 and EV3 to illustrate DHHC5 localisation.


Volcano plot of DHHC5 interactors from AP - MS analysis from HeLa cells co - expressing FLAG - DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin - mediated endocytosis , cell : cell adhesion and known substrates of DHHC5 .

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Volcano plot of DHHC5 interactors from AP-MS analysis from HeLa cells co-expressing FLAG-DHHC5 and WT Golga7b highlighting interacting proteins involved in clathrin-mediated endocytosis, cell:cell adhesion and known substrates of DHHC5.


Validation of novel DHHC5 interactors by co - IP : immunoblots of Dsg2from Hela cells co - expressing DHHC5 and either Wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of clathrin heavy chain from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * = p < 0 . 05 , ratio paired t - test , n = 3 . Co - IP immunoblots of ZO - 1 protein from Hela cells co - expressing DHHC5 and either wild - type ( WT ) or mutant ( m ) Golga7b and quantification normalised to input , * * = p < 0 . 01 ratio paired t - test , n = 3 .

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Validation of novel DHHC5 interactors by co-IP: immunoblots of Dsg2from Hela cells co-expressing DHHC5 and either Wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of clathrin heavy chain from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, *=p<0.05, ratio paired t-test, n=3. Co-IP immunoblots of ZO-1 protein from Hela cells co-expressing DHHC5 and either wild-type (WT) or mutant (m) Golga7b and quantification normalised to input, **=p<0.01 ratio paired t-test, n=3.


30 minute DMSO treatment of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) . 30 minute Dynasore treatment ( 25µM ) of HeLa cells co - expressing WT DHHC5 ( FLAG tagged ) and mutant Golga7b ( HA tagged ) .

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30 minute DMSO treatment of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged). 30 minute Dynasore treatment (25µM) of HeLa cells co-expressing WT DHHC5 (FLAG tagged) and mutant Golga7b (HA tagged).


Non - targeting control siRNA treatment ( 10nM ) of HeLa cells expressing mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment ( 10nM ) of HeLa cells that were subsequently transfected with mutant Golga7b ( HA tagged ) . Images are detecting endogenous DHHC5 and the HA tagged Golga7b .

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Non-targeting control siRNA treatment (10nM) of HeLa cells expressing mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b AP2µ siRNA treatment (10nM) of HeLa cells that were subsequently transfected with mutant Golga7b (HA tagged). Images are detecting endogenous DHHC5 and the HA tagged Golga7b.


Quantification of plasma membrane signal of DHHC5 in ( A and B ) . * * * * = p < 0 . 001 , unpaired t - test . N = 37 , + Dynasore . N = 28 , + DMSO .

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Quantification of plasma membrane signal of DHHC5 in (A and B). ****=p<0.001, unpaired t-test. N=37, +Dynasore. N=28, +DMSO.


Quantification of plasma membrane signal of DHHC5 in ( C and D ) . * * * * = p < 0 . 001 , unpaired t - test . All scale bars 10μm .

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Quantification of plasma membrane signal of DHHC5 in (C and D). ****=p<0.001, unpaired t-test. All scale bars 10μm.


Biotin endocytosis assays of HeLa cells with endogenous DHHC5 , over - expression of DHHC5 alone , co - expression of DHHC5 and WT Golga7b , co - expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C - terminal mutant . Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5 . The lack of signal in the pull - down of the DHHC5 C - terminal mutant or when DHHC5 and WT Golga7b are co - expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions . The robust signal in the pull - down of endocytosed proteins from cells co - expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition . N = 3 , * = p < 0 . 05 , paired t test "

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Biotin endocytosis assays of HeLa cells with endogenous DHHC5, over-expression of DHHC5 alone, co-expression of DHHC5 and WT Golga7b, co-expression of DHHC5 and mutant Golga7b or expression of the DHHC5 C-terminal mutant. Biotinylated cell surface proteins endocytosed within 15 minutes were purified using streptavidin resin and blots probed with an antibody to DHHC5. The lack of signal in the pull-down of the DHHC5 C-terminal mutant or when DHHC5 and WT Golga7b are co-expressed indicates that DHHC5 is stabilised at the plasma membrane in these conditions. The robust signal in the pull-down of endocytosed proteins from cells co-expressing DHHC5 and mutant Golga7b shows a high rate of endocytosis of DHHC5 in this condition. N=3, *=p<0.05, paired t test"


ABE purifications from HeLa cells treated with 10nM non - targeting siRNA ( nt siRNA ) or DHHC5 siRNA were immunoblotted for Dsg2 . Loss of the band in the + HA pull - down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5 .

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ABE purifications from HeLa cells treated with 10nM non-targeting siRNA (nt siRNA) or DHHC5 siRNA were immunoblotted for Dsg2. Loss of the band in the +HA pull-down in the DHHC5 siRNA condition shows that Dsg2 palmitoylation is dependent on DHHC5.


Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments .

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Confocal images of DHHC5 and Dsg2 in nt siRNA treated A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. Confocal images of DHHC5 and Dsg2 in DHHC5 depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments.


Confocal images of DHHC5 , Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments . See Appendix Figure S6 for quantification of data in panels B - D .

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Confocal images of DHHC5, Dsg2 and Golga7b in Golga7b depleted A431 cells three hours after calcium was reintroduced to the media in calcium switch experiments. See Appendix Figure S6 for quantification of data in panels B-D.


Immunoblots and quantification of Dsg2 pulled - down in cell surface biotinylation assays in nt siRNA , DHHC5 siRNA or Golga7b siRNA treated conditions . Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input . A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA . * =

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Immunoblots and quantification of Dsg2 pulled-down in cell surface biotinylation assays in nt siRNA, DHHC5 siRNA or Golga7b siRNA treated conditions. Quantification of the intensity of the DSG2 signal in the pulldown is normalised to input. A significant reduction of cell surface Dsg2 occurs when DHHC5 or Golga7b are depleted by siRNA. *=


Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium ( 5µM calcium ) conditions , with quantification of the intensity of the DSG2 signal in the pulldown normalised to input . There is a significant reduction in the Dsg2 signal in the + HA pull - down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition . * = p < 0 . 05 , paired t - test , N = 4 , error bars represent s . e . m . All scale bars 10μm .

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Immunoblot of an ABE purification for Dsg2 in cells with either normal media or low calcium (5µM calcium) conditions, with quantification of the intensity of the DSG2 signal in the pulldown normalised to input. There is a significant reduction in the Dsg2 signal in the +HA pull-down from low calcium cells showing that Dsg2 is significantly less palmitoylated in this condition. *=p<0.05, paired t-test, N=4, error bars represent s.e.m. All scale bars 10μm.


10x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5 ng / ml EGF treatment . 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed .

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10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5 ng/ml EGF treatment. 10x brightfield images of A431 cells treated with DHHC5 siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of DHHC5 knockdown and therefore a decrease in cell adhesion was observed.


10x brightfield images of A431 cells treated with non - targeting siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 ( left ) and after 48 hours ( right ) of 5ng / ml EGF treatment . Quantification of cell scattering of A431 cells , showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion , was observed .

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10x brightfield images of A431 cells treated with non-targeting siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. 10x brightfield images of A431 cells treated with Golga7b siRNA at time 0 (left) and after 48 hours (right) of 5ng/ml EGF treatment. Quantification of cell scattering of A431 cells, showing a significant increase in cell scatter after 48 hours of Golga7b knockdown and therefore a decrease in cell adhesion, was observed.


Plot showing the resistance of A431 cells from a trans - epithelial electrical resistance assay treated with either DHHC5 siRNA , Golga7b siRNA or non - targeting siRNA . A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion .

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Plot showing the resistance of A431 cells from a trans-epithelial electrical resistance assay treated with either DHHC5 siRNA, Golga7b siRNA or non-targeting siRNA. A significant reduction in TEER demonstrates a decrease in cell adhesion after both DHHC5 and Golga7b depletion.


B . Chromatin accessibility and p300 binding at differential ATAC - seq peaks visualized using K - means clustering .

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B. Chromatin accessibility and p300 binding at differential ATAC-seq peaks visualized using K-means clustering.


B . Boxplots showing differential gene expression ( AC versus DMZ ) and associated ATAC - seq signals ( Two biological replicates ) . The central band within the boxplot represents the median and the whiskers show 1 . 5 times the range between the first and third quartile ( IQR ) .

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B. Boxplots showing differential gene expression (AC versus DMZ) and associated ATAC-seq signals (Two biological replicates). The central band within the boxplot represents the median and the whiskers show 1.5 times the range between the first and third quartile (IQR).


F . Genomic tracks showing aggregated accessibility of single - cell ATAC - seq clusters at the t ( tbxt ) , tfap2a , gsc , ctcf , lhx1 and sox11 loci .

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F. Genomic tracks showing aggregated accessibility of single-cell ATAC-seq clusters at the t (tbxt), tfap2a, gsc, ctcf, lhx1 and sox11 loci.


C . Feature maps , showing the expression of selected genes in single cells ( cf . Appendix Fig . S2 ) . Color scale represents the gene expression value ( log1p - transformed counts per 10 , 000 unique reads ) for each cell for a given gene , from low ( grey ) to high ( orange ) .

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C. Feature maps, showing the expression of selected genes in single cells (cf. Appendix Fig. S2). Color scale represents the gene expression value (log1p-transformed counts per 10,000 unique reads) for each cell for a given gene, from low (grey) to high (orange).


A . Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1 , Foxb1 - Eomes , Eomes , Irx3 , Irx3 - Otx2 , Otx2 and Lhx8 .

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A. Heat map showing log2 fold expression changes of differentially expressed genes in AC tissues overexpressing Foxb1, Foxb1-Eomes, Eomes, Irx3, Irx3-Otx2, Otx2 and Lhx8.


C . Fold enrichment of genes with zygotically defined ( ZyD ) H3K4me3 at their promoter in AC with transcription factor overexpression . Asterisk indicates hypergeometric p - value < = 0 . 01 .

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C. Fold enrichment of genes with zygotically defined (ZyD) H3K4me3 at their promoter in AC with transcription factor overexpression. Asterisk indicates hypergeometric p-value <= 0.01.


( 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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

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(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 .

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(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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(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.


( 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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(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.


( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn and DSCR1

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(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 .

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(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 .

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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 .

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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 .

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(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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(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.


( b ) Suppression of Htt103Q toxicity in yeast by Gpx1 , Hyr1 ( Gpx3 ) and mGpx1 . The viability of the parental strain , Y258 , and of cells overexpressing yeast GPxs was determined using spotting assays . Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose ( GAL ) to induce the expression of Htt103Q and the indicated ORF .

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(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.


a ) Total GPx activity was measured in mGpx1 - overexpressing ( mGpx1 ) or control ( WT ) cell lines with ( induced ) or without ( uninduced ) Htt103Q expression . Overexpression of mGpx1 increases total GPx activity in PC12 cells ( * * * * P 0 . 0001 compared to uninduced controls ; n = 7 per condition ) .

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a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).


( b ) Quantification of caspase - 3 / 7 activation in response to Htt103Q expression in cells overexpressing mGpx1 . Overexpression of mGpx1 reduces Htt103Q - mediated caspase - 3 / 7 activation ( * * * * P 0 . 0001 ) . Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q ( n = 12 ( WT ) ; n = 9 ( monomeric red fluorescent protein ( mRFP ) ) ; n = 8 ( mGpx1 ) per condition ) .

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(b) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).


( c ) Effect of the GPx mimetic ebselen on Htt103Q - mediated caspase activation . A single 10 μM dose of ebselen significantly reduced caspase - 3 / 7 activation in Htt103Q - expressing cells ( * * * * P 0 . 0001 ) . Values represent the ratio of caspase - 3 / 7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q . Statistical analysis was performed using an unpaired , two - tailed Mann - Whitney test ( n = 12 per condition ) .

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(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).


( d ) mGpx1 overexpression ( * * * * P 0 . 0001 ) and ebselen treatment ( * * * * P 0 . 0001 ) significantly reduce Htt103Q - mediated ROS production ( n = 9 per condition ) . Unless stated otherwise , all statistical analyses were performed using one - way analysis of variance ( ANOVA ) with post - hoc tests . NS , not significant . * * P 0 . 01 . All data are shown as the mean ± s . e . m .

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(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.


( a ) Pseudopupil images from wild - type flies , Huntington ' s disease flies and Huntington ' s disease flies overexpressing mGpx1 at day 10 . In wild - type flies , seven rhabdomeres are visible per ommatidium . Scale bar , ∼ 10

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(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM


( b ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with and without pan - neural mGpx1 overexpression at days 1 ( n = 14 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) , 7 ( n = 7 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) and 10 ( n = 8 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) after eclosion . Significant effects of genotype ( F1 , 50 = 82 . 3 , P = 3 . 82 × 10 − 12 ) and age ( F2 , 50 = 94 . 3 , P = 1 . 06 × 10 − 17 ) were observed , as well as a significant interaction between genotype and age ( F2 , 50 = 7 . 76 , P = 0 . 00116 ) , indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies . Post - hoc Newman - Keuls comparisons showed significant rescue of neurodegeneration in mGpx1 - expressing flies at days 1 ( * P 0 . 05 ) , 7 ( * * * P 0 . 001 ) and 10 ( * * * P 0 . 001 ) . Browne - Forsythe analyses for equality of variance revealed a significant inequality of variance ( P = 0 . 02 ) due to a single group ( Htt93Q + mGpx1 , day 1 ) . The nonparametric Mann - Whitney test confirmed a difference at day 1 between the two groups ( P = 0 . 03 ) , and between Htt93Q + mGpx1 flies at days 1 and 7 ( P 0 . 0001 ) .

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(b) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).


( c ) Total locomotor activity was assessed as the average number of beam crossings per 30 - min bin per fly . Htt93Qex1 - expressing flies ( n = 29 ) have reduced locomotor activity compared to controls ( * * * P 0 . 001 ; n = 30 ( WT ( w118 ) ) ; n = 32 ( mGpx1 ) ) , which is restored by mGpx1 ( * * P 0 . 01 ; n = 32 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( d ) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7 . Flies expressing Htt93Qex1 driven by Pdf - GAL4 ( n = 16 brain hemispheres ) showed loss of s - LNVs compared to Canton S ( CS ) control flies ( n = 13 ) , which was ameliorated by coexpression of mGpx1 ( * * * * P 0 . 0001 ; n = 16 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( e ) Quantification of the average number of rhabdomeres per ommatidium in Huntington ' s disease flies with ebselen or vehicle ( DMSO ; n = 10 ) treatment at day 7 after eclosion and in newly emerged day 0 adult flies ( untreated control ; n = 10 ) . Significant treatment effects were observed , both with the 100 μM ( * * P 0 . 01 ; n = 9 ) and 300 μM ( * * * P 0 . 001 ; n = 9 ) doses of ebselen . All data are shown as the mean ± s . e . m .

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(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.


PC12 cells were treated with either 10 μM ebselen or 10 mM N - acetylcysteine ( NAc ) for 24 h , with the addition of 400 nM bafilomycin A1 for the final 4 h . Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h . ( a ) Autophagy was determined by measuring the amount of LC3 - II by immunoblot analysis . ( b ) Densitometric analysis of the bands was performed using ImageJ , and the amounts of LC3 - II are expressed relative to the untreated control . All statistical analyses were performed using one - way ANOVA with post - hoc tests ( * * * * P 0 . 0001 ) . All data are shown as the mean ± s . e . m . ; n = 7 per condition .

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PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.


A , Genome browser images of ChIP - seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


C , Mean ChIP - seq signal for USP22 , GCN5 , and ATXN7L3 surrounding gene promoters from B . Nonparametric Mann - Whitney analysis of 7 bins ( grey shade ) surrounding maximal peak in Thaps treated conditions are reported .

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C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.


A , HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs . Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours . Scale bar = 400 µm . B , Quantification of apoptotic populations as indicated by Cas3 / 7 fluorescence from A . Three independent experiments are represented as mean ± SEM , with significance calculated by Two - way ANOVA between conditions over time ( f ( 3 ) = 50 . 530 , p < 0 . 001 ) ; significant pairwise comparisons by Tukey ' s HSD post - hoc assessment are indicated by * . C

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A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C


A , HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22 . Cells were harvested and whole - cell lysate was subjected to immunoblotting with the indicated antibodies .

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

A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.


A , Genome browser images of ChIP - seq for Pol II and Ub - H2B before and after induction of ER stress , with and without USP22 knockdown , at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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

A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , USP22 - Bound genes . mRNA analyses of select Bound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 C , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , SAGA - Unbound genes . Nonparametric Mann - Whitney analyses of 7 bins ( grey shade ) surrounding TSS ( TSS ) and the midpoint of the profiles ( Body ) in Thaps treated conditions are reported . mRNA analysis of select Unbound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 005 .

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

B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.


C , ChIP - qPCR for total RNA Pol II , pSer5 Pol II , and pSer2 Pol II at ERP70 , GRP78 , and a gene desert ( Prom = gene promoter , Coding = downstream coding region - see Appendix Table S1 for primer sequences ) . Three independent experiments are represented as mean ± SEM . with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

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

C, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.


H , An aliquot of cells from D ‑ G were harvested for whole - cell lysate and subjected to immunoblotting with the indicated antibodies .

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

H, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.