Figure 5B . Western blot analysis of HCT116 / RIPK3 - 2xFV cells ( FL and ∆ C ) treated with 100 nM dimerizer . Blots are representative of two biological replicates . D . CXCL8 concentration in the culture media of HCT116 / RIPK3 - 2xFV cells ( WT and RHIMmut ) treated with 100 nM dimerizer for 24 h . In RIPK3 - RHIMmut three key residues , VQV , of the RHIM region was changed to AAAA . Data is plotted as mean with S . E . M . ( n = 5 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated time points . * * p = 0 . 0011 ; ns = not significant ( p = 0 . 9301 ) . Inset : Western blot analysis to determine RIPK3 expression levels in one biological replicate . Line indicates that image was cut and spliced to remove non - relevant lanes from the scanned blot . H . Relative mRNA levels of CXCL8 in HCT116 / RIPK3 - 2xFV cells pretreated with 100 nM CpA for 0 . 5 h before treatment with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO + dimerizer - treated condition . * * * * p < 0 . 0001 . ( H ) Cell lysates were analyzed by Western blotting for cIAP1 levels . Blots are representative of two biological replicates . J . Relative CXCL8 mRNA levels in WT or HOIP - knockout HCT116 / RIPK3 - 2xFV cells treated with 0 or 100 nM dimerizer for 3 h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference as indicated . * * * p = 0 . 0010 . Cell lysates were loaded for Western blot analysis . K . Relative CXCL8 mRNA levels in HCT116 / Tet - On - GFP - K63 - SUB / RIPK3 - 2xFV , HCT116 / Tet - On - GFP - M1 - SUB / RIPK3 - 2xFV and HCT116 / Tet - On - GFP / RIPK3 - 2xFV cells treated with 0 or 100 ng / mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p = 0 . 9997 ) ; * * p = 0 . 0030 ; * p = 0 . 0366 . Cells were analyzed by Western blotting for the inducible - expression levels of GFP ( control ) and GFP - SUBs . Blots are representative of three biological replicates . M . Relative CXCL8 mRNA levels in HCT116 / RIPK3 - 2xFV cells stably knocked down against mismatch ( shMM ) or two different sites of caspase - 8 ( shCASP8 - A and shCASP8 - B ) , pretreated with DMSO , 10 µM Nec1s , 10 µM GSK ' 872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . Two - way ANOVA and Tukey ' s multiple comparison tests were used to test for statistical differences between indicated conditions . * * * p = 0 . 0001 ; * * p = 0 . 0035 . Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase - 8 levels .

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 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 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 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 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

Figure 5B. Western blot analysis of HCT116 / RIPK3-2xFV cells (FL and ∆C) treated with 100 nM dimerizer. Blots are representative of two biological replicates.D. CXCL8 concentration in the culture media of HCT116 / RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data is plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. ** p = 0.0011; ns = not significant (p = 0.9301). Inset: Western blot analysis to determine RIPK3 expression levels in one biological replicate. Line indicates that image was cut and spliced to remove non-relevant lanes from the scanned blot.H. Relative mRNA levels of CXCL8 in HCT116 / RIPK3-2xFV cells pretreated with 100 nM CpA for 0.5 h before treatment with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO+dimerizer-treated condition. **** p < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1 levels. Blots are representative of two biological replicates.J. Relative CXCL8 mRNA levels in WT or HOIP-knockout HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. *** p = 0.0010. Cell lysates were loaded for Western blot analysis.K. Relative CXCL8 mRNA levels in HCT116 / Tet-On-GFP-K63-SUB / RIPK3-2xFV, HCT116 / Tet-On-GFP-M1-SUB / RIPK3-2xFV and HCT116 / Tet-On-GFP / RIPK3-2xFV cells treated with 0 or 100 ng/mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.9997); ** p = 0.0030; * p = 0.0366. Cells were analyzed by Western blotting for the inducible-expression levels of GFP (control) and GFP-SUBs. Blots are representative of three biological replicates.M. Relative CXCL8 mRNA levels in HCT116 / RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. *** p = 0.0001; ** p = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels.


Figure 6A . Western blot analysis of ZBP1 , RIPK1 , RIPK3 , MLKL levels in Calu - 3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence ( shMM ) , untreated ( NT ) or treated with 10 ng / mL IFNβ for 48 h . Blots are representative of two biological replicates .

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

Figure 6A. Western blot analysis of ZBP1, RIPK1, RIPK3, MLKL levels in Calu-3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence (shMM), untreated (NT) or treated with 10 ng/mL IFNβ for 48 h. Blots are representative of two biological replicates.


Figure 1A . BAZ2A expression is higher in ESC + 2i than in differentiated cells ( neural progenitors , NPC ) . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and NPC . Tubulin is shown as a protein loading control . B . BAZ2A associates with chromatin of ESC and NPC . Chromatin - bound ( Chrom . ) and soluble ( Sol . ) fractions of equivalent cell number of ESC + 2i and NPCs were analyzed by western blot for BAZ2A levels . Total , total protein . Tubulin and histones are shown as loading and fractionation control . C . siRNA - knockdown efficiency of BAZ2A shown by qRT - PCR and western blot . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to each ESC line . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * * < 0 . 0001 ) . Right panel . Western blot showing BAZ2A protein levels . Tubulin is shown as a protein loading control . D . BAZ2A knockdown affects proliferation of ESC + 2i but not of ESC + serum . Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 ; ns , non - significant ) . E . BAZ2A is expressed at similar levels in both ESC + 2i and ESC + serum . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and ESC + serum . Tubulin and histone H3 are shown as a protein loading controls . F . BAZ2A is required for the differentiation of ESC + 2i but not of ESC + serum . Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10 % serum and in the absence of LIF . G . Quantification of differentiated cells . Values represent relative number of differentiated cells from three independent experiments relative to control cells . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ; ns , non - significant ) . H . Western blot showing BAZ2A protein levels in two BAZ2A - KO ESC lines obtained via CRISPr / Cas9 directly in ESC + serum . PARP1 is shown as a protein loading control . I . Cell proliferation is not affected in BAZ2A - KO ESC + serum . Data represent relative cell numbers 2 days culture starting with the same number of cells . Average values of four independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns , non - significant ) . J . Representative images of wt and Baz2a - KO ESC + serum before and after transition in 2i conditions .

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

Figure 1A. BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control.B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.C. siRNA-knockdown efficiency of BAZ2A shown by qRT-PCR and western blot. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control.D. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant).E. BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls.F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF.G. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant).H. Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control.I. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant).J. Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions.


Figure 2A . Volcano plot showing fold change ( log2 values ) in transcript level of ESC + 2i and ESC + serum with two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Gene expression values of three replicates were averaged and selected for 1 . 5 fold changes and P < 0 . 05 . Statistical significance ( P - values ) was calculated using R package DEseq2 . B . Validation by qRT - PCR of genes regulated by BAZ2A in ESC + 2i but not in ESC + serum ( upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red ) . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . Data without P values are statistically non significant .

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 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

Figure 2A. Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2.B. Validation by qRT-PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P values are statistically non significant.


Figure 3A . Western blot showing equal BAZ2A levels in wt - ESC and F / H - BAZ2A ESC line containing FLAG - HA sequences at the N - terminus of both Baz2a alleles . Whole cell lysates from equivalent amounts of cells were analysed . SNF2H , and Tubulin are shown as protein loading controls . B . Anti - FLAG ChIP of wt - ESCs and F / H - BAZ2A - ESCs cultured in 2i or serum . Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 ) . C . Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3 . The grey rectangles highlighted some of BAZ2A associated regions . D . Pearson correlation heat map for the indicated ChIPseqs in ESC + 2i . BAZ2A ChIPseqs were performed with FLAG antibodies in F / H - BAZ2A - ESCs cultured in 2i or serum and with HA antibodies in F / H - BAZ2A - ESC + 2i . Data of H3K4me3 , H3K27ac and H3K27me3 are from this work . H3K36me3 , H3K9me3 and H3K4me1 values in ESC + 2i were taken from published data setsE . Boxplot showing the mean of normalized BAZ2A occupancies in ESC + 2i and ESC + serum at BAZ2A - bound regions . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . F . Differential BAZ2A - binding in 2i and serum correlates with H3K27ac occupancies . Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC + 2i and ESC + serum at all BAZ2A - bound regions and BAZ2A - bound regions only in 2i were calculated and their log2 - fold changes are plotted . H3K27ac in ESC + serum Statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . G . Representative images showing 2i - specific BAZ2A - bound regions and H3K27ac and H3K27me3 in ESC + 2i and ESC + serum . The grey box highlights strong TIP5 - bound regions and corresponding histone modifications . H . Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3 , depleted of H3K27ac and not bound by BAZ2A . I . Heat map profiles of H3K27me3 and H3K27ac at ± 5 kb from the TSS of genes differentially regulated by BAZ2A in ESC + 2i and random genes . Differentially regulated genes are shown as upregulated or downregulated in ESC + 2i upon BAZ2A knockdown . Data were ranked by H3K27me3 levels . Transcription start site ( TSS ) . J . Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over + / - 1kb of TSSs from upregulated , downregulated and random genes . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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 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 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 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 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 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 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 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 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 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

Figure 3A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.B. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01).C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions.D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data setsE. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications.H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A.I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS).J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


Figure 4A . Number of BAZ2A - bound regions in A and B compartments of ESC + serum . B . Hi - C contact matrices for a zoomed in region on chromosome 3 at 100 - kb resolution showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Low panels . HiC contacts of a zoomed region at 50 - kb resolution in ESC + serum , NPCs and CNs are shown . Data are from ( Bonev et al . , 2017 ) . HiC contacts were visualized using Juicebox C . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + serum . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . D . Long - range contacts marked by BAZ2A binding are specific for ESCs . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in all genome or in A compartments of ESC + serum , NPC and CN . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . E . Number of BAZ2A - bound regions in A and B compartments in ESC + 2i . F . BAZ2A - bound regions mark A subcompartments that are characterized by the strongest far - cis contacts in ESC + 2i . Boxplots indicating observed / expected contact values between BAZ2A - bound regions and loci not bound by BAZ2A at near - cis and far - cis contacts in all genome or in A compartments of ESC + 2i . Statistical significance ( P - values ) was calculated using the unpaired two - tailed t - test ( * * < 0 . 001 , * * * * < 0 . 0001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . G . BAZ2A binding correlates the best with eigenvector values . Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC + serum and ESC + 2i , respectively . H . Boxplots showing H3K4me1 and H3K27ac content of BAZ2A - bound regions and loci not bound by BAZ2A at far - cis contacts in ESC + 2i . Data from regions with lowest 10 % and top 10 % contact strength are shown . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 4A. Number of BAZ2A-bound regions in A and B compartments of ESC+serum. B. Hi-C contact matrices for a zoomed in region on chromosome 3 at 100-kb resolution showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Low panels. HiC contacts of a zoomed region at 50-kb resolution in ESC+serum, NPCs and CNs are shown. Data are from (Bonev et al., 2017). HiC contacts were visualized using Juicebox C. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+serum. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (****< 0.0001). D. Long-range contacts marked by BAZ2A binding are specific for ESCs. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in all genome or in A compartments of ESC+serum, NPC and CN. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.E. Number of BAZ2A-bound regions in A and B compartments in ESC+2i. F. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+2i. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments of ESC+2i. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (** < 0.001, ****< 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes. G. BAZ2A binding correlates the best with eigenvector values. Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC+serum and ESC+2i, respectively.H. Boxplots showing H3K4me1 and H3K27ac content of BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in ESC+2i. Data from regions with lowest 10% and top 10% contact strength are shown. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


Figure 5A . Numbers of ATAC peaks called separately in ESC + 2i and ESC + serum including their localization according to compartmentalization . B . Numbers of significant changed ATAC peaks in ESC + 2i vs . ESC + serum and their localization according to compartmentalization . Increased chromatin accessibility sites ( IAS ) , decreased accessibility sites ( DAS ) . C . Average profile of normalized ATAC read counts over differential peaks in ESC + 2i and ESC + serum changed ATAC peaks . The signal is plotted over + / - 1kb on the center of the ATAC peak . D . H3K27ac correlates with differential accessible sites between ESC + 2i and ESC + serum . Boxplot showing the mean values of normalized H3K27ac at ATAC peaks ( + / - 500bp ) that are increased and decreased in ESC + 2i compared to ESC + serum , respectively . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . E . BAZ2A depletion increases chromatin accessibility only in ESC + 2i . Numbers of significant changed ATAC peaks in ESC + 2i and ESC + serum upon BAZ2A knockdown and their localization according to compartmentalization . F . Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC + 2i and ESC + serum treated with siRNA - Baz2a and siRNA - control over changed ATAC peaks . The signal is plotted over + / - 1kb centered on the ATAC peak . G . The distances of increased ( IAS ) and decreased ( DAS ) chromatin accessibility sites in BAZ2A - depleted ESC + 2i to the nearest gene promoter ( + / - 5kb of TSS ) were calculated . Distances in bp to the nearest promoter of genes upregulated ( UP ) , downregulated ( DOWN ) or not affected ( NOT ) upon BAZ2A knockdown in ESC + 2i are plotted . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * < 0 . 01 ) . Error bars represent s . d . The mean is represented by a horizontal line . H . Genomic annotation of IAS and DAS in ESC + 2i depleted of BAZ2A perfomed with ChIPseekerI . Fold - enrichment relative to expected of regions with increased chromatin accessibility ( IAS ) and upregulated genes upon BAZ2A KD at sub - regions within compartment A and B ( center , inside and boundaries ) . Compartments smaller than 500 kb were excluded from the analysis . Statistical significance ( P - values ) was calculated with permutation test ( ns : non significant , * < 0 . 05 , * * * < 0 . 001 ) . J . BAZ2A - upregulated genes are enriched in the B compartment . Numbers of BAZ2A - regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC + 2i including their localization according to compartmentalization . K . Active histone marks increase in the B compartment upon BAZ2A knockdown . Boxplots indicating the log2 fold - changes of normalized H3K27ac and H3K4me3 read counts in ESC + 2i treated with siRNA - Baz2a and siRNA - control at boundaries and centers of A and B compartments . ) . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , * < 0 . 05 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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 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 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 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 B-PANEL_START 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 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 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

Figure 5A. Numbers of ATAC peaks called separately in ESC+2i and ESC+serum including their localization according to compartmentalization. B. Numbers of significant changed ATAC peaks in ESC+2i vs. ESC+serum and their localization according to compartmentalization. Increased chromatin accessibility sites (IAS), decreased accessibility sites (DAS). C. Average profile of normalized ATAC read counts over differential peaks in ESC+2i and ESC+serum changed ATAC peaks. The signal is plotted over +/- 1kb on the center of the ATAC peak. D. H3K27ac correlates with differential accessible sites between ESC+2i and ESC+serum. Boxplot showing the mean values of normalized H3K27ac at ATAC peaks (+/- 500bp) that are increased and decreased in ESC+2i compared to ESC+serum, respectively. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.E. BAZ2A depletion increases chromatin accessibility only in ESC+2i. Numbers of significant changed ATAC peaks in ESC+2i and ESC+serum upon BAZ2A knockdown and their localization according to compartmentalization. F. Average density plots of normalized ATAC read counts over differential ATAC peaks of ESC+2i and ESC+serum treated with siRNA-Baz2a and siRNA-control over changed ATAC peaks. The signal is plotted over +/- 1kb centered on the ATAC peak. G. The distances of increased (IAS) and decreased (DAS) chromatin accessibility sites in BAZ2A-depleted ESC+2i to the nearest gene promoter (+/- 5kb of TSS) were calculated. Distances in bp to the nearest promoter of genes upregulated (UP), downregulated (DOWN) or not affected (NOT) upon BAZ2A knockdown in ESC+2i are plotted. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (** < 0.01). Error bars represent s.d. The mean is represented by a horizontal line.H. Genomic annotation of IAS and DAS in ESC+2i depleted of BAZ2A perfomed with ChIPseekerI. Fold-enrichment relative to expected of regions with increased chromatin accessibility (IAS) and upregulated genes upon BAZ2A KD at sub-regions within compartment A and B (center, inside and boundaries). Compartments smaller than 500 kb were excluded from the analysis. Statistical significance (P-values) was calculated with permutation test (ns: non significant, * < 0.05, *** < 0.001).J. BAZ2A-upregulated genes are enriched in the B compartment. Numbers of BAZ2A-regulated genes upregulated and downregulated upon BAZ2A knockdown in ESC+2i including their localization according to compartmentalization.K. Active histone marks increase in the B compartment upon BAZ2A knockdown. Boxplots indicating the log2 fold-changes of normalized H3K27ac and H3K4me3 read counts in ESC+2i treated with siRNA-Baz2a and siRNA-control at boundaries and centers of A and B compartments. ). Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, * < 0.05). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


Figure 6A . Hi - C contact matrices for chromosome 1 of ESC + serum , ESC + 2i / siRNA - control and ESC + 2i / siRNA - Baz2a showing the presence of strong long - distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC + serum . Cells were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Eigenvector values are shown . Increase in far - cis contacts in ESC + 2i upon BAZ2A - KD are highlighted with a rectangle . B . Relative contact frequencies over genomic distances in ESCs treated with siRNA - control and siRNA - BAZ2A . Statistical significance ( P - values ) was calculated using the Kolmogorov - Smirnov test . C . Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments . Differences in eigenvector values were calculated from HiC data sets from ESC + 2i treated with siRNA - Baz2a and siRNA - control . Statistical significance ( P - values ) for the experiments was calculated using the unpaired two - tailed t - test ( * * * < 0 . 001 , ns : non significant ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6A. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.


Figure 7A . Western blot showing total levels of H3K27me3 in ESC + 2i transfected with siRNA - control and siRNA - Baz2a . Histone H3 is shown as a protein loading control . B . Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC + 2i and ESC + serum . ESCs were subjected to two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Mean of normalized H3K27me3 read counts in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a over all TSSs ( + / - 1kb ) were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted relative to the mean of normalized H3K27me3 occupancies . The red line represents the mean of H3K27me3 fold - changes . C . Representative images showing the H3K27me3 occupancy . D . Average density plot of ChIPseq read counts of H3K27me3 at - / + 5 kb from the TSS of refseq genes in ESCs treated with siRNA - control or siRNA - Baz2a Q1 : first quartile corresponding to sequences with high H3K27me3 content . Q2 - 4 : quartiles 2 - 4 corresponding to sequences with low H3K27me3 content . E . ChIP - qPCR of H3K27me3 in ESC + 2i and ESC + serum treated with siRNA - control or siRNA - Baz2a . Data were measured by qPCR and normalized to input and to Tshz1 value in control cells . Control represents an intergenic sequence that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . F . Boxplot showing changes in H3K27me3 in ESC + 2i upon BAZ2A at BAZ2A - regulated and random genes . Mean of normalized H3K27me3 read counts in ESC + 2i treated with siRNA - control or siRNA - Baz2a over TSSs ( + / - 1kb ) of upregulated , downregulated and random genes were calculated . Log2 - fold changes of ( siRNA - Baz2a / siRNA - control ) are plotted . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 and * * * < 0 . 001 ) . Box plots depict the minimum and maximum values . The mean is represented by a horizontal line within the boxes . G . Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC + 2i or ESC + serum transfected with siRNA - control or siRNA - Baz2a . Data were ranked by H3K27me3 content in the corresponding siRNA - control ESCs . Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels . H . H3K27me3 ChIP - qPCR in ESC + 2i ( top panel ) or ESC + serum ( bottom panel ) transfected with siRNA - control or siRNA - Baz2a . Values show H3K27me3 occupancy at four sequences ( # 1 - # 4 ) with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic sequence that does not contain H3K27me3 . Data were measured by qPCR and normalized to input and to sequence # 1 value in control cells . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 ) . I . Hi - C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster ( 10 - kb resolution in upper panels , 5 kb resolution in lower panels ) . Blue arrows ( A , B , and C ) indicate gain or increase of contacts in ESC + siRNA - Baz2a compared to control cells ( siRNA - control ) . The orange arrow ( D ) indicates loss or decrease in contacts .

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 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 B-PANEL_START 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 O 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 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 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

Figure 7A. Western blot showing total levels of H3K27me3 in ESC+2i transfected with siRNA-control and siRNA-Baz2a. Histone H3 is shown as a protein loading control.B. Scatterplot showing the changes in H3K27me3 upon BAZ2A depletion in ESC+2i and ESC+serum. ESCs were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Mean of normalized H3K27me3 read counts in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a over all TSSs (+/- 1kb) were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted relative to the mean of normalized H3K27me3 occupancies. The red line represents the mean of H3K27me3 fold-changes.C. Representative images showing the H3K27me3 occupancy.D. Average density plot of ChIPseq read counts of H3K27me3 at -/+ 5 kb from the TSS of refseq genes in ESCs treated with siRNA-control or siRNA-Baz2a Q1: first quartile corresponding to sequences with high H3K27me3 content. Q2-4: quartiles 2-4 corresponding to sequences with low H3K27me3 content.E. ChIP-qPCR of H3K27me3 in ESC+2i and ESC+serum treated with siRNA-control or siRNA-Baz2a. Data were measured by qPCR and normalized to input and to Tshz1 value in control cells. Control represents an intergenic sequence that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).F. Boxplot showing changes in H3K27me3 in ESC+2i upon BAZ2A at BAZ2A-regulated and random genes. Mean of normalized H3K27me3 read counts in ESC+2i treated with siRNA-control or siRNA-Baz2a over TSSs (+/- 1kb) of upregulated, downregulated and random genes were calculated. Log2-fold changes of (siRNA-Baz2a/siRNA-control) are plotted. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01 and *** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.G. Average density and heatmap profile of H3K27me3 transition state regions marked by CTCF sites in ESC+2i or ESC+serum transfected with siRNA-control or siRNA-Baz2a. Data were ranked by H3K27me3 content in the corresponding siRNA-control ESCs. Cluster 1 represents high to low H327me3 transition whereas Cluster 2 shows low to high H3K27me3 levels.H. H3K27me3 ChIP-qPCR in ESC+2i (top panel) or ESC+serum (bottom panel) transfected with siRNA-control or siRNA-Baz2a. Values show H3K27me3 occupancy at four sequences (#1-#4) with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic sequence that does not contain H3K27me3. Data were measured by qPCR and normalized to input and to sequence #1 value in control cells. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01).I. Hi-C contact matrices for a zoomed in region on chromosome 6 containing HoxA cluster (10-kb resolution in upper panels, 5 kb resolution in lower panels). Blue arrows (A, B, and C) indicate gain or increase of contacts in ESC+siRNA-Baz2a compared to control cells (siRNA-control). The orange arrow (D) indicates loss or decrease in contacts.


Figure 8B . BAZ2A - interacting proteins on chromatin . Mass spectrometry analysis of FLAG immunoprecipitates from wt and F / H - BAZ2A ESC + 2i . Values of peptide number are shown as the difference between peptides obtained in FLAG - IP of F / H - BAZ2A and wt ESC chromatin extracts . Data are from three independent experiments . The proteins found enriched in FLAG - IP of F / H - BAZ2A ESCs in all three experiments are shown . Further BAZ2A - interacting proteins identified in only two or one of the immunoprecipitation ( IP ) experiments Error bars represent s . d . C . Anti - HA IP of wt - and F / H - BAZ2A - ESC + 2i and ESC + serum . Western blot shows the interaction of BAZ2A with SNF2H , TOP2A and SMC1a . D . TOP2A and BAZ2A regulate gene expression in ESC + 2i via a shared pathway . qRT - PCR of genes regulated by BAZ2A in ESC + 2i ( left panel ) and in ESC + serum ( right panel ) upon TOP2A or BAZ2A knockdown by siRNA . ESCs were treated with the corresponding RNAs for 4 days . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) . E . Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A - regulated genes in ESC + 2i observed upon TOP2A or BAZ2A knockdown . ESCs were treated with the TOP2 inhibitor ICF - 193 for 24 hours . mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO . Upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) . F . ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC + 2i but not in ESC + serum . Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA - control . # 1 - # 4 indicated sequences with elevated H3K27me3 at CTCF boundary ( H3K27me3hi / CTCF ) . Control represents an intergenic region that does not contain H3K27me3 . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns : non significant , * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 8B. BAZ2A-interacting proteins on chromatin. Mass spectrometry analysis of FLAG immunoprecipitates from wt and F/H-BAZ2A ESC+2i. Values of peptide number are shown as the difference between peptides obtained in FLAG-IP of F/H-BAZ2A and wt ESC chromatin extracts. Data are from three independent experiments. The proteins found enriched in FLAG-IP of F/H-BAZ2A ESCs in all three experiments are shown. Further BAZ2A-interacting proteins identified in only two or one of the immunoprecipitation (IP) experiments Error bars represent s.d.C. Anti-HA IP of wt- and F/H-BAZ2A-ESC+2i and ESC+serum. Western blot shows the interaction of BAZ2A with SNF2H, TOP2A and SMC1a.D. TOP2A and BAZ2A regulate gene expression in ESC+2i via a shared pathway. qRT-PCR of genes regulated by BAZ2A in ESC+2i (left panel) and in ESC+serum (right panel) upon TOP2A or BAZ2A knockdown by siRNA. ESCs were treated with the corresponding RNAs for 4 days. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).E. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001).F. ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC+2i but not in ESC+serum. Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA-control. #1-#4 indicated sequences with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic region that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).


Figure 1B . Southern blotting confirms successful targeting . Introduction of an additional EcoRI site results in a 4 . 1 kb restriction fragment detectable by Southern for targeted ES cells ( A174T / + ) but not for parental DNA ( + / + ) . C . Capillary sequencing for Rnaseh2b + / + , Rnaseh2bA174T / + , and Rnaseh2bA174T / A174T DNA confirmed the presence of the introduced missense mutation . D . Mouse genotyping by multiplex PCR . Top : A 221 bp PCR product is present in wild - type mice ( + / + ) ; the Rnaseh2bA174T allele ( also ) give a 460 bp product . Bottom : Position of forward ( x ) and reverse primers ( y , z ) . E . Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T / A174T MEFs and RNASEH2BA177T / A177T LCLs . Representative of three independent experiments . G , H . RNase H2 enzyme activity is reduced in mouse and patient cells . ( G ) Enzyme activity for Rnaseh2bA174T / A174T MEFs and passage - matched Rnaseh2b + / + controls , against RNase H substrate ( RNA : DNA heteroduplex ) and RNase H2 - specific substrate , double stranded DNA with a single embedded ribonucleotide ( DRD : DNA ) . Mean activity for three independent cell lines , error bars , SEM . Enzymatic activity expressed relative to the average value of control MEFs . * * * = p < 0 . 001 , two - tailed t - test ( n = 3 Rnaseh2bA174T / A174T and n = 3 Rnaseh2b + / + control MEF lines ) . ( H ) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B - A177T mutation . Enzyme activity normalised to average activity of control lines . Three independent experiments , error bars SEM . * * * = p < 0 . 001 versus either control , two - tailed t - test .

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 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 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 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 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 O O O O O O O O O O O O O O O O O

Figure 1B. Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1 kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).C. Capillary sequencing for Rnaseh2b+/+, Rnaseh2bA174T/+, and Rnaseh2bA174T/A174T DNA confirmed the presence of the introduced missense mutation.D. Mouse genotyping by multiplex PCR. Top: A 221 bp PCR product is present in wild-type mice (+/+); the Rnaseh2bA174T allele (also) give a 460 bp product. Bottom: Position of forward (x) and reverse primers (y, z).E. Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2bA174T/A174T MEFs and RNASEH2BA177T/A177T LCLs. Representative of three independent experiments.G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.


Figure 2Figure 2 . Increased ISG expression in tissues from Rnaseh2bA174T / A174T miceA . Transcript levels of multiple ISGs are significantly elevated in heart . B . Transcript levels of a subset of ISGs are significantly increased in kidney . C . No ISG upregulation is evident in the brain . ISG transcript levels determined by RT - qPCR normalised to transcript levels of the housekeeping gene HPRT ( Oas1a was undetectable in brain ) . Each data point represents the mean of technical replicates of tissue RNA from a single mouse . n = 9 nine - month old Rnaseh2bA174T / A174T mice , n = 4 age - matched control wildtype C57BL / 6Jmice . Horizontal line , mean ; error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , two - tailed t - test .

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Figure 2Figure 2. Increased ISG expression in tissues from Rnaseh2bA174T/A174T miceA. Transcript levels of multiple ISGs are significantly elevated in heart.B. Transcript levels of a subset of ISGs are significantly increased in kidney.C. No ISG upregulation is evident in the brain. ISG transcript levels determined by RT-qPCR normalised to transcript levels of the housekeeping gene HPRT (Oas1a was undetectable in brain). Each data point represents the mean of technical replicates of tissue RNA from a single mouse. n=9 nine-month old Rnaseh2bA174T/A174T mice, n=4 age-matched control wildtype C57BL/6Jmice. Horizontal line, mean; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test.


Figure 3A . Validation of Rnaseh2b - / - MEF lines . RER activity ( DRD : DNA ) is undetectable in a Rnaseh2b - / - line consistent with complete inactivation of the Rnaseh2b gene ( Reijns et al , 2012 ) . Mean of three independent experiments ; error bars , SEM , * * * * = p < 0 . 0001 , two - tailed t - test . Rnaseh2b - / - and Rnaseh2b + / + control MEFs on a C57BL / 6 p53 - / - background . B . ISG transcript levels are increased in Rnaseh2b - / - MEFs . C . BeadArray transcript analysis ( Illumina ) detected induction of multiple ISGs , including the cytokines CXCL10 and CCL5 , but not other cytokine transcripts in Rnaseh2b - / - MEFs . Plotted , average fold enrichment versus p - value of significantly upregulated transcripts ( p < 0 . 05 , after multiple testing correction ) comparing two Rnaseh2b - / - MEF lines versus 4 Rnaseh2b + / + MEF lines . 17 out of 29 transcripts are ISGs . D . CXCL10 and CCL5 transcripts ( detected by RT - qPCR ) are significantly elevated . E . Increased CXCL10 and CCL5 protein ( detected by ELISA ) is secreted by Rnaseh2b - / - MEFs . ( B , D , E ) Mean data from three experiments for six independent Rnaseh2b - / - MEF lines versus four independent Rnaseh2b + / + MEF lines . Error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , Mann - Whitney U test .

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 B-PANEL_START 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 B-PANEL_START 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

Figure 3A. Validation of Rnaseh2b-/- MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b-/- line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, **** = p<0.0001, two-tailed t-test. Rnaseh2b-/- and Rnaseh2b+/+ control MEFs on a C57BL/6 p53-/- background.B. ISG transcript levels are increased in Rnaseh2b-/- MEFs.C. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b-/- MEFs. Plotted, average fold enrichment versus p-value of significantly upregulated transcripts (p<0.05, after multiple testing correction) comparing two Rnaseh2b-/- MEF lines versus 4 Rnaseh2b+/+ MEF lines. 17 out of 29 transcripts are ISGs.D. CXCL10 and CCL5 transcripts (detected by RT-qPCR) are significantly elevated.E. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b-/- MEFs. (B, D, E) Mean data from three experiments for six independent Rnaseh2b-/- MEF lines versus four independent Rnaseh2b+/+ MEF lines. Error bars, SEM. * = p<0.05, ** = p<0.01, Mann-Whitney U test.


Figure 4A . ISG activation and cytokine secretion in Rnaseh2b - / - MEFs is markedly impaired by cGAS or STING siRNA depletion . Upper left and lower panels : RT - qPCR of IFIT1 , cGAS and STING transcripts , 48 h after siRNA targeting luciferase ( control ) , cGAS or STING . Upper right panel : CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion . Concentration of CCL5 ( ELISA ) , normalised to luciferase siRNA control levels in each experiment . Mean from three independent experiments using one Rnaseh2b - / - MEF line ; error bars , SEM . * = p < 0 . 05 , * * = p < 0 . 01 , two - tailed t - test for RT - qPCR , one sample t - test for CCL5ELISA . B - D . ISG induction and cytokine secretion is abolished in Rnaseh2b - / - cGAS - / - MEFs . cGAS was targeted by CRISPR / Cas9 genome editing of a Rnaseh2b - / - MEF line to inactivate cGAS / STING signalling . In addition to sequence validation , functional inactivation of cGAS was confirmed in Rnaseh2b - / - cGas - / - CRISPR lines by the absence of CCL5 secretion in response to dsDNA ( Appendix Fig S4 ) . CCL5 ( B ) and CXCL10 ( C ) production , as well as ISG expression ( D ) was abrogated in Rnaseh2b - / - cGAS - / - clones , assessed by ELISA and RT - qPCR respectively . Four independent experiments , n = 2 Rnaseh2b - / - cGAS - / - clones , n = 4 Rnaseh2b - / - cGAS + / + clones , error bars , SEM of each experiment * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , two - tailed t - test . B - D . ISG induction and cytokine secretion is abolished in Rnaseh2b - / - cGAS - / - MEFs . cGAS was targeted by CRISPR / Cas9 genome editing of a Rnaseh2b - / - MEF line to inactivate cGAS / STING signalling . In addition to sequence validation , functional inactivation of cGAS was confirmed in Rnaseh2b - / - cGas - / - CRISPR lines by the absence of CCL5 secretion in response to dsDNA ( Appendix Fig S4 ) . CCL5 ( B ) and CXCL10 ( C ) production , as well as ISG expression ( D ) was abrogated in Rnaseh2b - / - cGAS - / - clones , assessed by ELISA and RT - qPCR respectively . Four independent experiments , n = 2 Rnaseh2b - / - cGAS - / - clones , n = 4 Rnaseh2b - / - cGAS + / + clones , error bars , SEM of each experiment * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , two - tailed t - test . E . ISG induction in Rnaseh2bA174T / A174T mice is STING dependent . RT - qPCR of RNA extracted from hearts from Sting + / + Rnaseh2bA174T / A174T ( n = 4 ) and Sting - / - Rnaseh2bA174T / A174T ( n = 6 ) three - month old mice . F . Absence of STING does not significantly decrease basal ISG expression . RT - qPCR of RNA extracted from hearts from Sting + / + ( n = 3 ) and Sting - / - ( n = 5 ) three - month old mice . ( E , F ) Each data point represents the mean of technical triplicates from one mouse . Error bars , SEM . * = p < 0 . 05 , Mann - Whitney U test .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 4A. ISG activation and cytokine secretion in Rnaseh2b-/- MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT-qPCR of IFIT1, cGAS and STING transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b-/- MEF line; error bars, SEM. * = p<0.05, ** = p<0.01, two-tailed t-test for RT-qPCR, one sample t-test for CCL5ELISA.B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.B-D. ISG induction and cytokine secretion is abolished in Rnaseh2b-/- cGAS-/- MEFs. cGAS was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b-/- MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b-/- cGas-/- CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D) was abrogated in Rnaseh2b-/- cGAS-/- clones, assessed by ELISA and RT-qPCR respectively. Four independent experiments, n=2 Rnaseh2b-/- cGAS-/- clones, n=4 Rnaseh2b-/- cGAS+/+ clones, error bars, SEM of each experiment * = p<0.05, ** = p<0.01, *** = p<0.001, two-tailed t-test.E. ISG induction in Rnaseh2bA174T/A174T mice is STING dependent. RT-qPCR of RNA extracted from hearts from Sting+/+ Rnaseh2bA174T/A174T (n=4) and Sting-/-Rnaseh2bA174T/A174T (n=6) three-month old mice.F. Absence of STING does not significantly decrease basal ISG expression. RT-qPCR of RNA extracted from hearts from Sting+/+ (n=3) and Sting-/- (n=5) three-month old mice. (E, F) Each data point represents the mean of technical triplicates from one mouse. Error bars, SEM. * = p<0.05, Mann-Whitney U test.


Figure 5A . Overexpression of RNase H1 in Rnaseh2b - / - cells restores RNase H activity against RNA : DNA hybrids to 81 ± 10 % of wildtype levels , while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA : DNA and DRD : DNA substrates ( RER ) . Rnaseh2b - / - MEFs were complemented with Rnaseh1 ( + H1 ) , Rnaseh2b ( + H2B ) or EGFP by retroviral infection . Mean of n = 3 independent experiments ± SEM . B , C . DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1 , measured by 53BP1 foci formation in detergent - extracted fixed cells . ( B ) Representative images ( scale bar , 10 µm ) . ( C ) At least 150 cells were counted for each cell line in three independent experiments . Mean ± SEM , * * * * = p < 0 . 0001 two - tailed t - test . B , C . DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1 , measured by 53BP1 foci formation in detergent - extracted fixed cells . ( B ) Representative images ( scale bar , 10 µm ) . ( C ) At least 150 cells were counted for each cell line in three independent experiments . Mean ± SEM , * * * * = p < 0 . 0001 two - tailed t - test . D - F . CCL5 ( D ) and CXCL10 production ( E ) , as well as ISG induction ( F ) in Rnaseh2b - / - MEFs are reduced close to wildtype levels ( Rnaseh2b + / + ) , by complementation with Rnaseh2b but not Rnaseh1 . Mean of n = 6 independent experiments ± SEM for complemented cells ; n = 3 independent experiments for Rnaseh2b - / - parental and Rnaseh2b + / + controls cells . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 two - tailed t - test indicates significantly reduced expression compared to Rnaseh2b - / - parental cells . D - F . CCL5 ( D ) and CXCL10 production ( E ) , as well as ISG induction ( F ) in Rnaseh2b - / - MEFs are reduced close to wildtype levels ( Rnaseh2b + / + ) , by complementation with Rnaseh2b but not Rnaseh1 . Mean of n = 6 independent experiments ± SEM for complemented cells ; n = 3 independent experiments for Rnaseh2b - / - parental and Rnaseh2b + / + controls cells . * = p < 0 . 05 , * * = p < 0 . 01 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 two - tailed t - test indicates significantly reduced expression compared to Rnaseh2b - / - parental cells .

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 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 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 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 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

Figure 5A. Overexpression of RNase H1 in Rnaseh2b-/- cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wildtype levels, while overexpression of RNase H2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b-/- MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n=3 independent experiments ± SEM.B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.B, C. DNA damage is reduced to wildtype levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent-extracted fixed cells. (B) Representative images (scale bar, 10 µm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, **** = p<0.0001 two-tailed t-test.D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.D-F. CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b-/- MEFs are reduced close to wildtype levels (Rnaseh2b+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n=6 independent experiments ± SEM for complemented cells; n=3 independent experiments for Rnaseh2b-/- parental and Rnaseh2b+/+ controls cells. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001 two-tailed t-test indicates significantly reduced expression compared to Rnaseh2b-/- parental cells.


Figure 1Representative confocal images of control ( CTL - 1 , 2 , 3 ) and DOA + patient fibroblasts carrying indicated monoallelic mutations imaged as described in A . Scale bar = 20μm . Passage number between P12 - 15 . Mitochondrial morphology quantification of B . Data represent mean ± SD of two independent experiments , ( 195 - 2496 cells per cell line ) , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significantRepresentative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , or non - targeting ( NT ) siRNAs for 72 hours and imaged as described in A . Scale bar = 20μm . Passage number between P12 - 14 . Mitochondrial morphology quantification of D . Data represent mean ± SD of three independent experiments , ( 3219 - 5857 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant . Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide ( CHX ) where indicated for 6 hours . Imaging as described in A . Scale bar = 20μm . Passage number between P14 - P15 . Mitochondrial morphology quantification of F . Data represent mean ± SD of two independent experiments , ( 879 - 4154 cells per cell line ) , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant .

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 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 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 1Representative confocal images of control (CTL-1, 2, 3) and DOA+ patient fibroblasts carrying indicated monoallelic mutations imaged as described in A. Scale bar=20μm. Passage number between P12-15. Mitochondrial morphology quantification of B. Data represent mean ± SD of two independent experiments, (195-2496 cells per cell line), One-way ANOVA; ** p < 0.01, ****p < 0.0001, ns; not significantRepresentative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, or non-targeting (NT) siRNAs for 72 hours and imaged as described in A. Scale bar=20μm. Passage number between P12-14. Mitochondrial morphology quantification of D. Data represent mean ± SD of three independent experiments, (3219-5857 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with 50μM cycloheximide (CHX) where indicated for 6 hours. Imaging as described in A. Scale bar=20μm. Passage number between P14-P15. Mitochondrial morphology quantification of F. Data represent mean ± SD of two independent experiments, (879-4154 cells per cell line), One-way ANOVA; ****p < 0.0001, ns; not significant.


Figure 3Representative confocal images of control ( CTL - 1 ) and OPA1S545R patient fibroblasts treated with OPA1 , DNM1L , PGS1 , and non - targeting ( NT ) siRNAs or indicated combinations for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 20μm . Passages number between P10 - 15 . Mitochondrial morphology quantification of ( A ) using control fibroblasts with fragmented ( OPA1 siRNA ) , normal ( non - targeting NT siRNA ) , and hypertubulated ( DNM1L siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 905 - 3695 cells per cell line ) , ( % fragmented ) ; ; * * * * p < 0 . 0001 , ns ; not significant . . Representative confocal images of wild type ( WT ) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Mitochondrial morphology quantification of ( C ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) ground truth training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 6613 - 8758 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . Representative confocal images of WT , Opa1Crispr MEFs complemented with pLenti - Opa1 , Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti - Pgs1 by lentiviral delivery . Live imaging of mitochondria ( mitoYFP , green ) and nuclei ( NucBlue , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( E ) using WT MEFs treated with Opa1 siRNA ( fragmented ) , NT siRNA ( normal ) , or Dnm1l siRNA ( hypertubulated ) training sets . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 691 - 3990 cells per cell line ) , ( % fragmented ) ; * * * * p < 0 . 0001 , ns ; not significant . .

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 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

Figure 3Representative confocal images of control (CTL-1) and OPA1S545R patient fibroblasts treated with OPA1, DNM1L, PGS1, and non-targeting (NT) siRNAs or indicated combinations for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=20μm. Passages number between P10-15. Mitochondrial morphology quantification of (A) using control fibroblasts with fragmented (OPA1 siRNA), normal (non-targeting NT siRNA), and hypertubulated (DNM1L siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (905-3695 cells per cell line), (% fragmented); ; ****p < 0.0001, ns; not significant..Representative confocal images of wild type (WT) and Opa1Crispr MEFs treated with NT or Pgs1 siRNA for 72 hours. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Mitochondrial morphology quantification of (C) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) ground truth training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (6613-8758 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant.Representative confocal images of WT, Opa1Crispr MEFs complemented with pLenti-Opa1, Opa1CrisprPgs1Crispr MEFs and Pgs1Crispr MEFs complemented with pLenti-Pgs1 by lentiviral delivery. Live imaging of mitochondria (mitoYFP, green) and nuclei (NucBlue, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (E) using WT MEFs treated with Opa1 siRNA (fragmented), NT siRNA (normal), or Dnm1l siRNA (hypertubulated) training sets. Data represent mean ± SD of three independent experiments, One-way ANOVA (691-3990 cells per cell line), (% fragmented) ; ****p < 0.0001, ns; not significant..


Figure 4Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m - chlorophenyl hydrazine ( CCCP ) for the indicated time points . Images were captured every hour for 18 hours . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of three independent experiments , ( 131 - 426 cells per cell line ) , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant . Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion ( SiMH ) with 10μM cycloheximide ( CHX ) for the indicated time points . Images were captured every hour for 9 hours . Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h ( fragmented ) , untreated ( normal ) , or treated with 10μM CHX for 9h ( hypertubular ) training sets . Data represent mean ± SD of four independent experiments , ( 155 - 745 cells per cell line ) , One - way ANOVA .

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

Figure 4Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 hours. Scale bar=10μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of three independent experiments, (131-426 cells per cell line), One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 hours. Mitochondrial morphology quantification of using WT MEFs treated with 5μM CCCP for 18h (fragmented), untreated (normal), or treated with 10μM CHX for 9h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155-745 cells per cell line), One-way ANOVA.


Figure 5Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( B ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubular ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of three independent experiments , One - way ANOVA ( 726 - 4236 cells per cell line ) , ( % fragmented ) ; * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant . Representative confocal micrographs of MEFs WT , Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours . Mitochondria ( anti - TOM40 , green ) and nuclei ( DAPI , blue ) . Scale bar = 10μm . Supervised ML mitochondrial morphology quantification of ( G ) using WT MEFs with fragmented ( Opa1 siRNA ) , normal ( non - targetting NT siRNA ) , and hypertubulated ( Dnm1l siRNA ) mitochondria . Data represent mean ± SD of > 3 independent experiments , ( 3096 - 7238 cells per cell line ) , One - way ANOVA ( % fragmented ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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 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

Figure 5Representative confocal micrographs of MEFs WT and Opa1Crispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (B) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubular (Dnm1l siRNA) mitochondria. Data represent mean ± SD of three independent experiments, One-way ANOVA (726-4236 cells per cell line), (% fragmented); *** p < 0.001, ****p < 0.0001, ns; not significant.Representative confocal micrographs of MEFs WT, Pgs1Crispr and Dnm1lCrispr MEFs treated with indicated siRNAs for 72 hours. Mitochondria (anti-TOM40, green) and nuclei (DAPI, blue). Scale bar=10μm. Supervised ML mitochondrial morphology quantification of (G) using WT MEFs with fragmented (Opa1 siRNA), normal (non-targetting NT siRNA), and hypertubulated (Dnm1l siRNA) mitochondria. Data represent mean ± SD of >3 independent experiments, (3096-7238 cells per cell line), One-way ANOVA (% fragmented) ; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Figure 6Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs . Scale bar = 200nm . Quantification of ( C ) of mitochondrial ultrastructure ; outer membrane / inner membrane ration ( IMM / OMM ) and cristae number per mitochondrion . Violin plot of > 50 mitochondria per cell line , One - way ANOVA ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant .

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

Figure 6Representative transmission electron micrographs of MEFs of the indicated genotypes showing loss of lamellar cristae in Opa1Crispr and Opa1CrisprPgs1Crispr MEFs. Scale bar=200nm. Quantification of (C) of mitochondrial ultrastructure; outer membrane/inner membrane ration (IMM/OMM) and cristae number per mitochondrion. Violin plot of >50 mitochondria per cell line, One-way ANOVA; *p < 0.05, ** p < 0.01, ****p < 0.0001, ns; not significant.


Figure 7mtDNA content in MEFs from ( F ) was quantified by amplification of MTTL1 , 16S and MT - ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * * * p < 0 . 0001 , ns ; not significant . mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1 , 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * * p < 0 . 0001 , ns ; not significant . ( G ) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS - PAGE ( horizontal line denotes separate membranes ) , immunoblotted with indicated antibodies and quantified by densitometry ( H ) . Data represent mean ± SD of three independent experiments , One - way ANOVA ; * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , ns ; not significant .

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 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

Figure 7mtDNA content in MEFs from (F) was quantified by amplification of MTTL1, 16S and MT-ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; ****p < 0.0001, ns; not significant. mtDNA content in WT and mutant MEFs treated with indicated siRNAs for 72 hours was quantified by amplification of MTTL1, 16S and ND1 genes relative to the GAPDH nuclear gene in MEFs. Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ****p < 0.0001, ns; not significant.(G) Equal amounts of protein extracted from WT and mutant MEFs were separated by SDS-PAGE (horizontal line denotes separate membranes), immunoblotted with indicated antibodies and quantified by densitometry (H). Data represent mean ± SD of three independent experiments, One-way ANOVA; **p < 0.01, ***p < 0.001, ****p < 0.0001, ns; not significant.