Figure 4Expression of YAP1 and downstream effectors in SCP - 1 cells transduced with FUS ‑ DDIT3 or EV . One of at least three independent experiments with similar results is shown . Expression of YAP1 and downstream effectors in cytoplasmic ( yellow ) and nuclear ( blue ) fractions from SCP - 1 cells transduced with FUS ‑ DDIT3 or EV . One of at least two independent experiments with similar results is shown . YAP1 - responsive luciferase activity in SCP - 1 cells transduced with FUS ‑ DDIT3 . Relative luciferase activity is displayed as fold change relative to control . Error bars represent the mean ± SD of three independent experiments , unpaired t - test . Localization of FUS - DDIT3 and YAP1 in MLS 1765 - 92 . Nuclei were counterstained with DAPI . The original magnification was x63 , and images were zoomed in four times for co - localization analysis . The correlation between red and green fluorescence was determined by Pearson coefficient analysis ( square , area for signal acquisition ) . Co ‑ IP of transiently expressed FUS ‑ DDIT3 and YAP1 from HEK293T cells . V5 - tagged YAP1 was pulled down using an anti - V5 antibody , and interacting proteins were detected by immunoblotting . One of at least two independent experiments with similar results is shown . Co - IP of endogenous FUS - DDIT3 and YAP1 from MLS 1765 - 92 cells . FUS - DDIT3 was pulled down using an anti - DDIT3 antibody , and interacting proteins were detected by immunoblotting . One of at least two independent experiments with similar results is shown .

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 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 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 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

Figure 4Expression of YAP1 and downstream effectors in SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least three independent experiments with similar results is shown.Expression of YAP1 and downstream effectors in cytoplasmic (yellow) and nuclear (blue) fractions from SCP-1 cells transduced with FUS‑DDIT3 or EV. One of at least two independent experiments with similar results is shown.YAP1-responsive luciferase activity in SCP-1 cells transduced with FUS‑DDIT3. Relative luciferase activity is displayed as fold change relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.Localization of FUS-DDIT3 and YAP1 in MLS 1765-92. Nuclei were counterstained with DAPI. The original magnification was x63, and images were zoomed in four times for co-localization analysis. The correlation between red and green fluorescence was determined by Pearson coefficient analysis (square, area for signal acquisition).Co‑IP of transiently expressed FUS‑DDIT3 and YAP1 from HEK293T cells. V5-tagged YAP1 was pulled down using an anti-V5 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.Co-IP of endogenous FUS-DDIT3 and YAP1 from MLS 1765-92 cells. FUS-DDIT3 was pulled down using an anti-DDIT3 antibody, and interacting proteins were detected by immunoblotting. One of at least two independent experiments with similar results is shown.


Figure 5Viability and proliferation of MLS 402 ‑ 91 , MLS 1765 ‑ 92 , and MLS 2645 - 94 cells cultured in the presence of verteporfin . Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate . Flow cytometric analysis of apoptosis ( cleaved PARP ) and mitotic fraction ( phosphorylated histone H3S10 ) in MLS cell lines cultured in the presence of 0 . 25 µM Verteporfin . One of two independent experiments with similar results is shown . Expression of total YAP1 and downstream effectors ( FOXM1 and PLK1 ) in MLS cell lines treated with 0 . 5 or 1 µM verteporfin for 15 hours . One of at least three independent experiments with similar results is shown . YAP1 - responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin . Relative luciferase activity is displayed relative to control . Error bars represent the mean ± SD of three independent experiments , unpaired t - test . Tumor formation on chicken CAM of MLS cell lines following shRNA - mediated YAP1 knockdown . The number of tumors is given above each bar , Fisher exact test ; ns , not significant . Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin . Shown are tumor volumes and representative photographs of tumors .

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 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 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 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 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

Figure 5Viability and proliferation of MLS 402‑91, MLS 1765‑92, and MLS 2645-94 cells cultured in the presence of verteporfin. Error bars represent the mean ± SEM of one representative experiment performed in quintuplicate.Flow cytometric analysis of apoptosis (cleaved PARP) and mitotic fraction (phosphorylated histone H3S10) in MLS cell lines cultured in the presence of 0.25 µM Verteporfin. One of two independent experiments with similar results is shown.Expression of total YAP1 and downstream effectors (FOXM1 and PLK1) in MLS cell lines treated with 0.5 or 1 µM verteporfin for 15 hours. One of at least three independent experiments with similar results is shown.YAP1-responsive luciferase activity in MLS cell lines transfected with a constitutively active YAP1S127A mutant and treated with 1 µM verteporfin. Relative luciferase activity is displayed relative to control. Error bars represent the mean ± SD of three independent experiments, unpaired t-test.Tumor formation on chicken CAM of MLS cell lines following shRNA-mediated YAP1 knockdown. The number of tumors is given above each bar, Fisher exact test; ns, not significant.Tumor growth on chicken CAM of MLS cell lines following treatment with 1 µM verteporfin. Shown are tumor volumes and representative photographs of tumors.


Figure 1C . Cell cycle fractions inferred from Cycleflow in IFN - β - treated NSCs . n = 3 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

Figure 1C. Cell cycle fractions inferred from Cycleflow in IFN-β-treated NSCs. n = 3 biological replicates.


Figure 2C , D . Ribo - Seq results depicting translation efficiency as the interaction of log2 fold changes ( LFC ) between footprints mapped to the CDS , referred to as " CDS counts " , and total RNA after 2h or 14h IFN - β treatment . FDR < 10 % , LR - Test in DESeq2 . Genes with a p value < 0 . 1 after FDR correction are highlighted . n = 4 biological replicates . I . Representative WB and phosphorylation levels quantification ( log2FC ) of the mTOR - related proteins ribosomal protein S6 kinase ( S6K ) , ribosomal protein S6 ( S6 ) and eIF4E - binding protein 1 ( 4E - BP1 ) from NSCs treated with IFN - β and normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - 4E - BP1S65 * ( p = 0 . 021 ) , p - S6S235 / 236 & p - S6S240 / 244 * * * ( p < 0 . 001 ) , p - S6KT389 * * ( p = 0 . 004 ) . n = biological replicates specified . p ≡ p - value .

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 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 2C,D. Ribo-Seq results depicting translation efficiency as the interaction of log2 fold changes (LFC) between footprints mapped to the CDS, referred to as "CDS counts", and total RNA after 2h or 14h IFN-β treatment. FDR < 10%, LR-Test in DESeq2. Genes with a p value<0.1 after FDR correction are highlighted. n = 4 biological replicates.I. Representative WB and phosphorylation levels quantification (log2FC) of the mTOR-related proteins ribosomal protein S6 kinase (S6K), ribosomal protein S6 (S6) and eIF4E-binding protein 1 (4E-BP1) from NSCs treated with IFN-β and normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-4E-BP1S65 * (p=0.021), p-S6S235/236 & p-S6S240/244 ***(p<0.001), p-S6KT389 **(p=0.004). n = biological replicates specified. p ≡ p-value.


Figure 3A . Representative WB image and quantification ( log2FC ) of p - TSC2Thr1462 & p - AktThr308 from IFNβ - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . One - way ANOVA for the biphasic response test p - TSC2T1462 * * * ( p < 0 . 001 ) , p - AktT308 * * ( p = 0 . 003 ) . n = 4 biological replicates . Data information : p ≡ p - value . C . Representative WB images and quantifications ( log2FC ) of p - eIF2αS51 in IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - eIF2αS51 * * ( p = 0 . 003724 ) . n = 4 biological replicates . Data information : p ≡ p - value . E . Representative WB image and quantifications ( log2FC ) of p - Rb1Ser780 from IFN - β - treated NSCs normalized to control ( t = 0h ) . Bars represent the mean value . Spearman ' s rank correlation test . p - Rb1S780 * * * ( p = 5 . 37e - 06 ) . n = 3 biological replicates . Data information : p ≡ p - value . I , J . Luciferase activity controlled by the upstream 5 ' UTR fragment from Sox2 ( WT and mutant ) , Rps21 ( WT and mutant ) and Actb . Treatments were applied for 12h . Data is normalized to vehicle and is represented as mean ± SD . n = 6 biological replicates . Two - way ANOVA with Tukey ' s multiple comparisons test ( p - value specified ) . Data information : p ≡ p - value .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative WB image and quantification (log2FC) of p-TSC2Thr1462 & p-AktThr308 from IFNβ-treated NSCs normalized to control (t=0h). Bars represent the mean value. One-way ANOVA for the biphasic response test p-TSC2T1462 ***(p<0.001), p-AktT308 **(p=0.003). n = 4 biological replicates. Data information: p ≡ p-value.C. Representative WB images and quantifications (log2FC) of p-eIF2αS51 in IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-eIF2αS51 **(p=0.003724). n = 4 biological replicates. Data information: p ≡ p-value.E. Representative WB image and quantifications (log2FC) of p-Rb1Ser780 from IFN-β-treated NSCs normalized to control (t=0h). Bars represent the mean value. Spearman's rank correlation test. p-Rb1S780 ***(p=5.37e-06). n = 3 biological replicates. Data information: p ≡ p-value.I,J. Luciferase activity controlled by the upstream 5'UTR fragment from Sox2 (WT and mutant), Rps21 (WT and mutant) and Actb. Treatments were applied for 12h. Data is normalized to vehicle and is represented as mean ± SD. n = 6 biological replicates. Two-way ANOVA with Tukey's multiple comparisons test (p-value specified). Data information: p ≡ p-value.


Figure 4B . vSVZ niche FACS quantifications of total NSCs ( Total NSCs ) & immunofluorescence quantification of aNSCs ( Fraction of aNSCs ) in WT or IFNAGR KO mice at 2 , 7 and 22 months old ( MO ) . Data from individual mice is plotted . Inferred dynamics are depicted as a blue ( WT ) or red ( IFNAGR KO ) line over time . Sample sizes : WT total at 60 days : 8 mice , WT total at 210 days : 3 mice , WT total at 660 days : 3 mice , WT fraction aNSCs at 60 days : 3 mice , WT fraction aNSCs at 660 days : 4 mice , IFNAGRKO total at 60 days : 8 mice , IFNAGRKO total at 180 days : 3 mice , IFNAGRKO total at 660 days : 3 mice , IFNAGRKO fraction aNSCs at 60 days : 3 mice , IFNAGRKO fraction aNSCs at 660 days : 3 mice .

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

Figure 4B. vSVZ niche FACS quantifications of total NSCs (Total NSCs) & immunofluorescence quantification of aNSCs (Fraction of aNSCs) in WT or IFNAGR KO mice at 2, 7 and 22 months old (MO). Data from individual mice is plotted. Inferred dynamics are depicted as a blue (WT) or red (IFNAGR KO) line over time. Sample sizes: WT total at 60 days: 8 mice, WT total at 210 days: 3 mice, WT total at 660 days: 3 mice, WT fraction aNSCs at 60 days: 3 mice, WT fraction aNSCs at 660 days: 4 mice, IFNAGRKO total at 60 days: 8 mice, IFNAGRKO total at 180 days: 3 mice, IFNAGRKO total at 660 days: 3 mice, IFNAGRKO fraction aNSCs at 60 days: 3 mice, IFNAGRKO fraction aNSCs at 660 days: 3 mice.


figf1a , Impairment of two ATG - conjugation systems ( Atg12 and LC3 ) in the Atg7 - deficient brain . Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7 , Atg5 and LC3 . Actin was used as a loading control . Data shown are representative of three separate experiments . b , Kaplan - Meier survival curves of Atg7flox / + ; nestin - Cre ( n = 41 ) and Atg7flox / flox ; nestin - Cre ( n = 26 ) mice over 28 weeks . c , Abnormal limb - clasping reflexes in Atg7flox / flox ; nestin - Cre mice at P28 . When lifted by the tail , Atg7flox / + ; nestin - Cre mice behave normally , extending their hind limbs and bodies . In contrast , Atg7flox / flox ; nestin - Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs . d , Movement ataxia in Atg7flox / flox ; nestin - Cre mice at P28 . Motor coordination was tested using a rotarod assay . Atg7flox / + ; nestin - Cre ( n = 5 ) and Atg7flox / flox ; nestin - Cre ( n = 5 ) mice were placed on a rod rotating at 20 r . p . m . , and the time spent on the rod was counted . Data show mean ± s . d . * , P 0 . 01 ( Students t - test ) . There was no significant sex difference in survival rate and onset - stage of abnormal mice - clasping and tremor in miceflox / flox ; nestin - Cre mice .

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 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 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

figf1a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in the Atg7-deficient brain. Brain homogenates from P28 mice were immunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was used as a loading control. Data shown are representative of three separate experiments.b, Kaplan-Meier survival curves of Atg7flox/+; nestin-Cre (n = 41) and Atg7flox/flox; nestin-Cre (n = 26) mice over 28 weeks.c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28. When lifted by the tail, Atg7flox/+; nestin-Cre mice behave normally, extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cre mice bend their legs towards their trunk or tighten their back limbs to their bodies and anterior limbs.d, Movement ataxia in Atg7flox/flox; nestin-Cre mice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/+; nestin-Cre (n = 5) and Atg7flox/flox; nestin-Cre (n = 5) mice were placed on a rod rotating at 20 r.p.m., and the time spent on the rod was counted. Data show mean ± s.d. *, P 0.01 (Students t-test). There was no significant sex difference in survival rate and onset-stage of abnormal mice-clasping and tremor in miceflox/flox; nestin-Cre mice.


figf2a - f , Histological analyses of Atg7flox / + ; nestin - Cre ( left ) and Atg7flox / flox ; nestin - Cre ( right ) cerebral cortex at P56 . Cryosections were stained with HE ( a - d ) or immunostained for the glial marker GFAP ( e , f ) . Boxed areas in a and b are magnified in c and d , respectively . Arrows in c point to large pyramidal neurons in the cerebral cortex . g - j , Histological analysis of Atg7flox / + ; nestin - Cre ( left ) and Atg7flox / flox ; nestin - Cre ( right ) cerebellum at P56 . Cryosections were stained with HE ( g , h ) or immunostained for the Purkinje marker calbindin ( i , j ) . Arrows in g and h indicate cerebellar Purkinje cells . k - p , TUNEL staining of the cerebral cortex ( k , l ) and cerebellum ( n , o ) at P56 in Atg7flox / + ; nestin - Cre ( k , n ) and Atg7flox / flox ; nestin - Cre ( l , o ) sections . TUNEL - positive cells are indicated with arrows and shown as higher magnification images beneath panels l , n and o . Histograms show the average number ( ± s . d . ) of TUNEL - positive cells in ten sections for three animals of each genotype ( m , p ) . * , P 0 . 05 ( t - test ) . Scale bars , 1 mm ( a , b ) , 100 µm ( c - j ) , 250 µm ( k , l , n , o ) . We observed no sex difference in brain morphology or neuronal loss in Atg7flox / flox ; nestin - Cre mice .

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 O O O O O 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

figf2a-f, Histological analyses of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebral cortex at P56. Cryosections were stained with HE (a-d) or immunostained for the glial marker GFAP (e, f). Boxed areas in a and b are magnified in c and d, respectively. Arrows in c point to large pyramidal neurons in the cerebral cortex.g-j, Histological analysis of Atg7flox/+; nestin-Cre (left) and Atg7flox/flox; nestin-Cre (right) cerebellum at P56. Cryosections were stained with HE (g, h) or immunostained for the Purkinje marker calbindin (i, j). Arrows in g and h indicate cerebellar Purkinje cells.k-p, TUNEL staining of the cerebral cortex (k, l) and cerebellum (n, o) at P56 in Atg7flox/+; nestin-Cre (k, n) and Atg7flox/flox; nestin-Cre (l, o) sections. TUNEL-positive cells are indicated with arrows and shown as higher magnification images beneath panels l, n and o. Histograms show the average number (± s.d.) of TUNEL-positive cells in ten sections for three animals of each genotype (m, p). *, P 0.05 (t-test). Scale bars, 1 mm (a, b), 100 µm (c-j), 250 µm (k, l, n, o). We observed no sex difference in brain morphology or neuronal loss in Atg7flox/flox; nestin-Cre mice.


figf3a - l , The presence of ubiquitin - positive dots was examined by immunohistochemistry in several regions including cerebral cortex ( a , b ) , cerebellum ( c , d ) , hippocampus ( e , f ) , hypothalamus ( g , h ) , amygdala ( i , j ) and pontine nuclei ( k , l ) of Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice . Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants . Scale bars , 50 µm . m , n , Electron micrographs of the brain of Atg7flox / flox ; nestin - Cre mice . Inclusion bodies ( arrows ) were often observed in Atg7flox / flox ; nestin - Cre hypothalamus . The boxed region in m is shown in n . Inclusion bodies were not detected in Atg7flox / + ; nestin - Cre brain ( data not shown ) . Scale bars , 5 µm ( m ) , 1 µm ( n ) . o , Immunoelectron micrograph of ubiquitin in a representative Atg7flox / flox ; nestin - Cre hypothalamus . N , nucleus . Scale bar , 1 µm . p , q , Immunohistochemical detection of ubiquitin - positive inclusions in the cerebral cortex ( p ) and hypothalamus ( q ) at P9 , P18 and P56 . Brain sections of each genotype at the indicated ages were immunostained with an anti - ubiquitin antibody . Ubiquitin - positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox / flox ; nestin - Cre mice . Scale bars , 50 µm .

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

figf3a-l, The presence of ubiquitin-positive dots was examined by immunohistochemistry in several regions including cerebral cortex (a, b), cerebellum (c, d), hippocampus (e, f), hypothalamus (g, h), amygdala (i, j) and pontine nuclei (k, l) of Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice. Note the presence of numerous ubiquitin dots in the amygdala and hypothalamus of the representative mutants. Scale bars, 50 µm.m, n, Electron micrographs of the brain of Atg7flox/flox; nestin-Cre mice. Inclusion bodies (arrows) were often observed in Atg7flox/flox; nestin-Cre hypothalamus. The boxed region in m is shown in n. Inclusion bodies were not detected in Atg7flox/+; nestin-Cre brain (data not shown). Scale bars, 5 µm (m), 1 µm (n). o, Immunoelectron micrograph of ubiquitin in a representative Atg7flox/flox; nestin-Cre hypothalamus. N, nucleus. Scale bar, 1 µm.p, q, Immunohistochemical detection of ubiquitin-positive inclusions in the cerebral cortex (p) and hypothalamus (q) at P9, P18 and P56. Brain sections of each genotype at the indicated ages were immunostained with an anti-ubiquitin antibody. Ubiquitin-positive inclusions appeared at P18 and became larger with ageing in the brain of Atg7flox/flox; nestin-Cre mice. Scale bars, 50 µm.


figf4a , Increase in ubiquitinated proteins in Atg7flox / flox ; nestin - Cre mouse brain over time . Homogenates of P9 , P18 and P56 brains from Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice were immunoblotted with an anti - ubiquitin antibody . An anti - actin antibody was used as a loading control . Data shown are representative of three separate experiments . We observed no sex difference in the accumulation of ubiquitin in Atg7flox / flox ; nestin - Cre mice . b , Peptide hydrolysis activity of 20S and 26Sproteasomes . Homogenates from P28Atg7flox / + ; nestin - Cre ( blue ) and Atg7flox / flox ; nestin - Cre ( pink ) brains were fractionated by glyceroldensity gradient centrifugation ( 10 - 40 % glycerol from fraction 1 to fraction 30 ) . Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc - LLVY - AMC as a substrate in the absence ( top ) or presence ( bottom ) of 0 . 05 % SDS . The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads . Note that whereas 26Sproteasomes exist in active forms in tissues , 20Sproteasomes are latent and are activated artificially by a low concentration of SDS . c , ATP - dependent degradation of [ 35S ] - labelled ODC . Degradation of [ 35S ] - labelled ODC was assayed using crude extracts from P28 Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains . The experiment was repeated three times , and values represent mean ± s . d . In the above assays , there were no significant differences between Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre mice . d , Immunoblot analysis of 26S proteasome components . Homogenates from P28 Atg7flox / flox , Atg7flox / + ; nestin - Cre and Atg7flox / flox ; nestin - Cre brains were immunoblotted with antibodies against the indicated proteins . Data shown are representative of three separate experiments . There was no change in proteasome status for the different genotypes .

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

figf4a, Increase in ubiquitinated proteins in Atg7flox/flox; nestin-Cre mouse brain over time. Homogenates of P9, P18 and P56 brains from Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice were immunoblotted with an anti-ubiquitin antibody. An anti-actin antibody was used as a loading control. Data shown are representative of three separate experiments. We observed no sex difference in the accumulation of ubiquitin in Atg7flox/flox; nestin-Cre mice.b, Peptide hydrolysis activity of 20S and 26Sproteasomes. Homogenates from P28Atg7flox/+; nestin-Cre (blue) and Atg7flox/flox; nestin-Cre (pink) brains were fractionated by glyceroldensity gradient centrifugation (10-40% glycerol from fraction 1 to fraction 30). Aliquots from each fraction were used for the assay of chymotryptic activity of proteasomes using Suc-LLVY-AMC as a substrate in the absence (top) or presence (bottom) of 0.05% SDS. The sedimenting positions of 20S and 26Sproteasomes are indicated with arrowheads. Note that whereas 26Sproteasomes exist in active forms in tissues, 20Sproteasomes are latent and are activated artificially by a low concentration of SDS.c, ATP-dependent degradation of [35S]-labelled ODC. Degradation of [35S]-labelled ODC was assayed using crude extracts from P28 Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains. The experiment was repeated three times, and values represent mean ± s.d. In the above assays, there were no significant differences between Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre mice.d, Immunoblot analysis of 26S proteasome components. Homogenates from P28 Atg7flox/flox, Atg7flox/+; nestin-Cre and Atg7flox/flox; nestin-Cre brains were immunoblotted with antibodies against the indicated proteins. Data shown are representative of three separate experiments. There was no change in proteasome status for the different genotypes.


Figure 1A - C . Immunodetection of BES1 - CFP from heat - treated plants . Plants pre - grown at 21 ° C were ( A ) exposed for 60 minutes to the indicated temperatures , ( B ) to 45 ° C for the indicated period of time , or ( C ) to 45 ° C and , following sampling after 30 minutes ( lane 2 ) and 60 minutes ( lane 3 ) , returned to 21 ° C for 3 hours ( lane 4 ) . CBB , Coomassie Brilliant Blue stained gel . D . Subcellular localization of BES1 - CFP in roots of 7 - day - old plants after heat shock . 35S : BES1 - CFP seedlings were grown at 21 ° C and either left untreated or exposed for 60 minutes to 45 ° C . Left : representative photos . Right : quantification of the nuclear / cytoplasmic signal . Data show the mean ± SD . n = 25 . The asterisks indicate significant differences to the untreated plants by Student ' s t - test ( * * * P ≤ 0 . 001 ) . F . Heat stress resistance of bes1 - 1 , bes1 - 2 and bes1 - D as compared to wild - type . For basal resistance assays ( top charts ) plants were directly exposed to 45 ° C for the indicated time . For acquired resistance ( bottom charts ) , plants were primed with the respective temperatures for 120 minutes before transfer to 45 ° C for 150 minutes . Data show the mean ± SE . n = 7 . The asterisks indicate significant differences compared to wild - type by Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 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 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 O O O O O O O O O O O O O O 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 1A-C. Immunodetection of BES1-CFP from heat-treated plants. Plants pre-grown at 21°C were (A) exposed for 60 minutes to the indicated temperatures, (B) to 45°C for the indicated period of time, or (C) to 45°C and, following sampling after 30 minutes (lane 2) and 60 minutes (lane 3), returned to 21°C for 3 hours (lane 4). CBB, Coomassie Brilliant Blue stained gel.D. Subcellular localization of BES1-CFP in roots of 7-day-old plants after heat shock. 35S:BES1-CFP seedlings were grown at 21°C and either left untreated or exposed for 60 minutes to 45°C. Left: representative photos. Right: quantification of the nuclear/cytoplasmic signal. Data show the mean ±SD. n=25. The asterisks indicate significant differences to the untreated plants by Student's t-test (***P≤0.001).F. Heat stress resistance of bes1-1, bes1-2 and bes1-D as compared to wild-type. For basal resistance assays (top charts) plants were directly exposed to 45°C for the indicated time. For acquired resistance (bottom charts), plants were primed with the respective temperatures for 120 minutes before transfer to 45°C for 150 minutes. Data show the mean ±SE. n=7. The asterisks indicate significant differences compared to wild-type by Student's t-test (*P≤0.05; **P≤0.01; ***P≤0.001).


Figure 2B . Immunodetection of BES1 - CFP from heat - stressed plants , treated with epiBL or BRZ . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 1 μM 24 - epiBL or 5 μM BRZ and then heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes ; lane 4 : 45 ° C for 60 minutes and recovery at 21 ° C for 180 minutes . C , D . Immunodetection of BES1 - CFP from heat - treated bri1 - 1 or det2 - 1 plants . Left : phenotypes of 10 - day - old plants of all lines as compared to wild - type Col - 0 ( wt ) . Right : Immunoblots from the lines grown and treated as in B . F . Immunodetection of BES1 - CFP from heat - treated bin2 - 1 plants .

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 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

Figure 2B. Immunodetection of BES1-CFP from heat-stressed plants, treated with epiBL or BRZ. 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 1 μM 24-epiBL or 5 μM BRZ and then heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes; lane 4: 45°C for 60 minutes and recovery at 21°C for 180 minutes.C, D. Immunodetection of BES1-CFP from heat-treated bri1-1 or det2-1 plants. Left: phenotypes of 10-day-old plants of all lines as compared to wild-type Col-0 (wt). Right: Immunoblots from the lines grown and treated as in B.F. Immunodetection of BES1-CFP from heat-treated bin2-1 plants.


Figure 3A . Immunodetection of BES1 - CFP from heat - stressed plants , treated with ABA or Fluridone ( Flu ) . 35S : BES1 - CFP plants were treated for 24 hours with DMSO ( control ) , 5 μM ABA or 10 μM Fluridone and heat - stressed as follows : lane 1 : untreated control ; lane 2 : 45 ° C for 30 minutes ; lane 3 : 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue . C . Immunodetection of native BES1 from heat - stressed and epiBL - treated abitM plants . abitM and wild - type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45 ° C for 60 minutes . The non - phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue .

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 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

Figure 3A. Immunodetection of BES1-CFP from heat-stressed plants, treated with ABA or Fluridone (Flu). 35S:BES1-CFP plants were treated for 24 hours with DMSO (control), 5 μM ABA or 10 μM Fluridone and heat-stressed as follows: lane 1: untreated control; lane 2: 45°C for 30 minutes; lane 3: 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.C. Immunodetection of native BES1 from heat-stressed and epiBL-treated abitM plants. abitM and wild-type plants were grown on ½ MS medium either containing DMSO as a control or 1 μM epiBL and either left untreated or exposed to 45°C for 60 minutes. The non-phosphorylated BES1 bands were quantified with ImageJ and the obtained values are shown in blue.


Figure 4C . ChIP to determine BES1 - CFP enrichment on the promoters of HSP70 . 3 , HSP70 . 4 and HSP90 . 1 following heat stress . 35S : BES1 - CFP and wild - type plants grown at 21 ° C were either left untreated or treated with 45 ° C for 60 minutes . BES1 - CFP was immuno - precipitated with α - GFP beads . Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated . TA3 ( 70 . 3p and 90 . 1p ) or 5s rRNA ( 70 . 4p ) were used for normalization . UBQ5 served as an internal control . Data show the mean ± SE . n = 3 biological replicates measured in 3 technical repeats . Significant differences as compared to wild - type at 21 ° C , calculated by Student ' s t - test , are shown ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4C. ChIP to determine BES1-CFP enrichment on the promoters of HSP70.3, HSP70.4 and HSP90.1 following heat stress. 35S:BES1-CFP and wild-type plants grown at 21°C were either left untreated or treated with 45°C for 60 minutes. BES1-CFP was immuno-precipitated with α-GFP beads. Fragments were quantified in the precipitates with qPCR and the ratios of samples with antibody to without antibody were calculated. TA3 (70.3p and 90.1p) or 5s rRNA (70.4p) were used for normalization. UBQ5 served as an internal control. Data show the mean ±SE. n=3 biological replicates measured in 3 technical repeats. Significant differences as compared to wild-type at 21°C, calculated by Student's t-test, are shown (*P≤0.05; **P≤0.01; ***P≤0.001).


Figure 5A . EMSAs showing in vitro DNA interaction of recombinant BES1 and / or HSFA1 with HSE - containing fragments of the HSP70 . 3 and HSP90 . 1 promoters . Unlabeled competitor fragments with functional HSEs ( C1 + HSE ) or mutated versions ( C2 - HSE ) were used for competition studies in 100x molar access ( lanes 6 and 7 ) . B . BiFC assays showing cells co - transformed with nYFP - BES1 and cYFP - HSFA1a . The fusion proteins were co - expressed in N . benthamiana leaves and fluorescence was visualized . Two nuclei are highlighted and shown in a 6 - fold magnification . Scale bar = 20 μm . C . LUC assays in protoplasts of wild - type , bes1 - D or the hsfA1qM plants . LUC reporter constructs , driven by 2 kb of the HSP70 . 3 , HSP70 . 4 and HSP90 . 1 promoters , were tested either alone ( 0 , with empty effector plasmid ) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed . Data show the mean ± SD . n = 5 . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s 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 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 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

Figure 5A. EMSAs showing in vitro DNA interaction of recombinant BES1 and/or HSFA1 with HSE-containing fragments of the HSP70.3 and HSP90.1 promoters. Unlabeled competitor fragments with functional HSEs (C1 +HSE) or mutated versions (C2 -HSE) were used for competition studies in 100x molar access (lanes 6 and 7).B. BiFC assays showing cells co-transformed with nYFP-BES1 and cYFP-HSFA1a. The fusion proteins were co-expressed in N. benthamiana leaves and fluorescence was visualized. Two nuclei are highlighted and shown in a 6-fold magnification. Scale bar=20 μm.C. LUC assays in protoplasts of wild-type, bes1-D or the hsfA1qM plants. LUC reporter constructs, driven by 2 kb of the HSP70.3, HSP70.4 and HSP90.1 promoters, were tested either alone (0, with empty effector plasmid) or with BES1 or HSFA1a as effectors and LUC transactivation was analyzed. Data show the mean ±SD. n=5. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


Figure 6A , B . Constitutive and BR - responsive growth of bes1 - DxhsfA1qM seedlings . Hypocotyl elongation of 7 - day - old dark - grown ( A ) and light - grown ( B ) seedlings on medium containing either 1 μM epi - BL , 1 μM BRZ or DMSO as a control ( c ) . Left : Mean and SD of 30 plants . Significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined with a Student ' s t - test . Right : photos of representative plants . C . Expression of BR - regulated genes in bes1 - DxhsfA1qM , as compared to its parents and respective wild - types analyzed by qPCRs . Data show the mean ± SD . n = 3 biological repeats , each measured in 3 technical replicates , normalized to UBC . Statistically significant difference at P ≤ 0 . 05 of results is indicated with different letters and was determined by Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 6A, B. Constitutive and BR-responsive growth of bes1-DxhsfA1qM seedlings. Hypocotyl elongation of 7-day-old dark-grown (A) and light-grown (B) seedlings on medium containing either 1 μM epi-BL, 1 μM BRZ or DMSO as a control (c). Left: Mean and SD of 30 plants. Significant difference at P≤0.05 of results is indicated with different letters and was determined with a Student's t-test. Right: photos of representative plants.C. Expression of BR-regulated genes in bes1-DxhsfA1qM, as compared to its parents and respective wild-types analyzed by qPCRs. Data show the mean ±SD. n=3 biological repeats, each measured in 3 technical replicates, normalized to UBC. Statistically significant difference at P≤0.05 of results is indicated with different letters and was determined by Student's t-test.


Figure 1Figure 1 . CHCHD10 resides in the same complex than Mitofilin , CHCHD3 and CHCHD6 . A . Second dimension of the Blue - Native ( BN ) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin , CHCHD3 and CHCHD6 in isolated mitochondria from mouse brainB . BN - PAGE of the MICOS and OXPHOS complexes in control ( C ) and patient ( P1 , P2 ) fibroblasts . Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody ( CI ) , 70kDa subunit antibody ( CII ) , core I antibody ( CIII ) and cytochrome c oxidase subunit I antibody ( CIV ) . MICOS complex was detected with an anti - Mitofilin antibody . C . Second dimension of the BN - PAGE showing that the steady - state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts . D . Analysis of OXPHOS supercomplexes in control and patient fibroblasts . BN - PAGE from isolated mitochondria permeabilized with 6g / g ( w / v ) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies . SC , supercomplexes I + III2 + IVn . The dividing lines correspond to gels sections visible in the raw data file .

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

Figure 1Figure 1. CHCHD10 resides in the same complex than Mitofilin, CHCHD3 and CHCHD6. A. Second dimension of the Blue-Native (BN) PAGE showing that CHCHD10 migrates with the MICOS protein subunits Mitofilin, CHCHD3 and CHCHD6 in isolated mitochondria from mouse brainB. BN-PAGE of the MICOS and OXPHOS complexes in control (C) and patient (P1, P2) fibroblasts. Complexes I to IV of OXPHOS were detected with the 39kDa subunit antibody (CI), 70kDa subunit antibody (CII), core I antibody (CIII) and cytochrome c oxidase subunit I antibody (CIV). MICOS complex was detected with an anti-Mitofilin antibody.C. Second dimension of the BN-PAGE showing that the steady-state levels of assembled CHCHD10 in MICOS complex are decreased in patient fibroblasts.D. Analysis of OXPHOS supercomplexes in control and patient fibroblasts. BN-PAGE from isolated mitochondria permeabilized with 6g/g (w/v) of digitonin immunoblotted on PVDF membrane and incubated with the indicated antibodies. SC, supercomplexes I+III2+IVn. The dividing lines correspond to gels sections visible in the raw data file.


Figure 2Figure 2 . Interaction of CHCHD10 with components of MICOS complex . A . Co - immunonoprecipitation ( IP ) of endogenous mitofilin , CHCHD10 and CHCHD3 in control and patient fibroblasts ( 1mg of total extracts were used for each IP ( left lower panel ) and 200µg were used for the input ( left upper panel ) ) . The same results were found in P2 fibroblasts ( not shown ) . Reverse co - IP experiment in HeLa cells with an antibody against CHCHD10 ( right lower panel ) . 1mg of total extracts were used for each IP and 200µg were used for the input ( right upper panel ) . The dividing lines correspond to gels sections visible in the raw data file . B . Duolink proximity ligation assay between Mitofilin and CHCHD10 ( upper panels ) and , CHCHD10 and CHCHD6 ( lower panels ) in control and patient fibroblasts observed by confocal microscopy . Mitochondria were stained with Mitotracker . Duolink spots per cell were quantified for 30 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) . Scale bar = 10 µm .

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 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

Figure 2Figure 2. Interaction of CHCHD10 with components of MICOS complex. A. Co-immunonoprecipitation (IP) of endogenous mitofilin, CHCHD10 and CHCHD3 in control and patient fibroblasts (1mg of total extracts were used for each IP (left lower panel) and 200µg were used for the input (left upper panel)). The same results were found in P2 fibroblasts (not shown). Reverse co-IP experiment in HeLa cells with an antibody against CHCHD10 (right lower panel). 1mg of total extracts were used for each IP and 200µg were used for the input (right upper panel). The dividing lines correspond to gels sections visible in the raw data file.B. Duolink proximity ligation assay between Mitofilin and CHCHD10 (upper panels) and, CHCHD10 and CHCHD6 (lower panels) in control and patient fibroblasts observed by confocal microscopy. Mitochondria were stained with Mitotracker. Duolink spots per cell were quantified for 30 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001). Scale bar = 10 µm.


Figure 3Figure 3 . Decrease of nucleoid number without mtDNA depletion in patient fibroblasts . A . In control and patient fibroblasts , nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker . Image analysis was performed by confocal microscopy . Scale bar : 10 µm . B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied fibroblast cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p < 0 . 001 ) . Nucleoid number ( B ) : patient 1 versus control * * * : p = 0 . 0001 , patient 2 versus control * * * : p = 0 . 001 . Nucleoid size ( C ) : patient 1 versus control * * * : p = 0 . 0001 . D . Mitochondrial DNA content in control and patientfibroblasts was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nuclear DNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

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 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

Figure 3Figure 3. Decrease of nucleoid number without mtDNA depletion in patient fibroblasts. A. In control and patient fibroblasts, nucleoids were labeled with an antibody against DNA and mitochondria were stained with Mitotracker. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied fibroblast cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): extremely significant (***: p<0.001). Nucleoid number (B): patient 1 versus control ***: p=0.0001, patient 2 versus control ***: p=0.001. Nucleoid size (C): patient 1 versus control ***: p=0.0001. D. Mitochondrial DNA content in control and patientfibroblasts was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nuclear DNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 4Figure 4 . Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles . A . Transfections were performed with empty vector ( EV ) or vectors encoding wild - type CHCHD10 - FLAG ( WT ) , CHCHD10 - FLAG ( S59L ) or CHCHD10 - FLAG ( P34S ) mutants . Mitochondria were stained with Mitotracker . Cells overexpressing wild - type and mutant CHCHD10 were labeled with FLAG antibodies . Nucleoids were visualized with an antiserum against DNA . Image analysis was performed by confocal microscopy . Scale bar : 10 µm . B - C . The average number ( B ) and size ( C ) of nucleoids , labeled with antibodies against DNA shown in A , were quantified for 35 randomly - selected individual cells per each studied cell line from 2 independent experiments . Differences between the cell lines were analyzed by Student ' s t - test ( two - sided ) : very significant ( * * : p = 0 . 0280 ) or extremely significant ( * * * : p = 0 . 0001 ) . D . Mitochondrial DNA content was quantified by real - time PCR . Data were expressed as ratio between mtDNA and nDNA concentration . Results represent the mean of relative PCR ± SD of 3 independent experiments . Statistical analysis were performed by Student ' s t - test ( two - sided ) .

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 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

Figure 4Figure 4. Decrease of nucleoid number without mtDNA depletion in HeLa cells overexpressing the CHCHD10S59L and CHCHD10P34S alleles. A. Transfections were performed with empty vector (EV) or vectors encoding wild-type CHCHD10-FLAG (WT), CHCHD10-FLAG (S59L) or CHCHD10-FLAG (P34S) mutants. Mitochondria were stained with Mitotracker. Cells overexpressing wild-type and mutant CHCHD10 were labeled with FLAG antibodies. Nucleoids were visualized with an antiserum against DNA. Image analysis was performed by confocal microscopy. Scale bar: 10 µm.B-C. The average number (B) and size (C) of nucleoids, labeled with antibodies against DNA shown in A, were quantified for 35 randomly-selected individual cells per each studied cell line from 2 independent experiments. Differences between the cell lines were analyzed by Student's t-test (two-sided): very significant (**: p=0.0280) or extremely significant (***: p=0.0001). D. Mitochondrial DNA content was quantified by real-time PCR. Data were expressed as ratio between mtDNA and nDNA concentration. Results represent the mean of relative PCR ± SD of 3 independent experiments. Statistical analysis were performed by Student's t-test (two-sided).


Figure 5Figure 5 . Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress . A . Total extracts from control ( C ) fibroblasts and intact isolated mitochondria from control and patient fibroblasts ( P1 , P2 ) were analyzed by immunoblotting using antibodies against PCNA ( nuclear protein ) , GAPDH ( cytosolic protein ) or SMAC ( mitochondrial intermembrane space protein ) ( upper panel ) . Mitochondria from patient and control fibroblasts were incubated with NP - 40 and separated into pellets ( P ) and supernatants ( S ) . The fractions of each extraction were subjected to western blot analysis . VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins , respectively ( middle panel ) . Ratio of mtDNA amplified from supernatant / mtDNA amplified from pellet , by qPCR , was quantified in control and patient fibroblasts ( lower panel ) . B . Determination of the mtDNA copy number after H2O2 treatment . mtDNA / nDNA values in control , patient 1 and patient 2 fibroblasts . mtDNA : mitochondrial DNA , nDNA : nuclear DNA . C . mtDNA repair after H2O2 treatment . A long - range PCR was used to evaluate the oxidative damage , induced by H2O2 treatment , in mtDNA . The relative PCR amplification of a 15 . 6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment . mtDNA repair activity in control , patient 1 and patient 2 relative PCR . B - C . Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times . Untreated control cultures were incubated in serum - free medium alone . Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed . Values were normalized to untreated cells and differences were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) , very significant ( * * : 0 . 01 > p > 0 . 001 ) . Patient 1 versus control : * : p = 0 . 015 ( recovery 1h ) , * : p = 0 . 022 ( recovery 2h ) , * * : p = 0 . 003 ( recovery 4h ) . Patient 2 versus control : * : p = 0 . 0230 ( recovery 2h ) , * : p = 0 . 041 ( recovery 4h ) . NT : cells not treated with

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 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 O O O O O O O O O

Figure 5Figure 5. Nucleoid disorganization in patient fibroblasts leading to a defect in mtDNA repair under conditions of oxidative stress. A. Total extracts from control (C) fibroblasts and intact isolated mitochondria from control and patient fibroblasts (P1, P2) were analyzed by immunoblotting using antibodies against PCNA (nuclear protein), GAPDH (cytosolic protein) or SMAC (mitochondrial intermembrane space protein) (upper panel). Mitochondria from patient and control fibroblasts were incubated with NP-40 and separated into pellets (P) and supernatants (S). The fractions of each extraction were subjected to western blot analysis. VDAC and SMAC were used to identify behaviors of well defined mitochondrial proteins that are integral membrane and soluble proteins, respectively (middle panel). Ratio of mtDNA amplified from supernatant/mtDNA amplified from pellet, by qPCR, was quantified in control and patient fibroblasts (lower panel).B. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 relative PCR. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2


Figure 6Figure 6 . No autophagy inhibition ( A - C ) and no mitophagy defect ( D - E ) in patient fibroblasts . A . Immunoblot of LC3B - I / - II expression in fibroblasts of patient 2 ( 1 , 2 ) , patient 1 ( 3 , 4 ) and control individual ( 5 , 6 ) in absence ( - ) or presence ( + ) of chloroquine ( CLQ ) . B . Quantitative analysis of LC3B - II / β - tubulin ratio from immunoblots ( n = 3 ) . C . Quantitative analysis of LC3B - II + CLQ / LC3B - II - CLQ ratio from immunoblots ( n = 3 ) . D . Confocal images of fibroblasts derived from a control , patient 1 and patient 2 . Boxed regions are enlarged . Scale bar : 20µm . E . Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol ( FL lyso / FL mito ) . Statistical analysis were performed on mean ± SEM using Student ' s t - test ( two - sided ) .

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

Figure 6Figure 6. No autophagy inhibition (A-C) and no mitophagy defect (D-E) in patient fibroblasts. A. Immunoblot of LC3B-I/-II expression in fibroblasts of patient 2 (1,2), patient 1 (3,4) and control individual (5,6) in absence (-) or presence (+) of chloroquine (CLQ). B. Quantitative analysis of LC3B-II/β-tubulin ratio from immunoblots (n=3). C. Quantitative analysis of LC3B-II + CLQ/ LC3B-II - CLQ ratio from immunoblots (n=3).D. Confocal images of fibroblasts derived from a control, patient 1 and patient 2. Boxed regions are enlarged. Scale bar: 20µm. E. Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol (FL lyso/FL mito ). Statistical analysis were performed on mean ± SEM using Student's t-test (two-sided).


Figure 7Figure 7 . CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts . A . Control ( C1 , C2 ) and patient ( P1 , P2 ) fibroblasts were treated with 1 μM of staurosporine ( STS ) for 16 h . Cell death was determined by flow cytometry using Annexin V / DAPI staining . Three independent experiments were performed per condition with two points analyzed per experiment . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : p = 0 . 0116 ) or extremely significant ( * * * : p = 0 . 0001 ) . B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control ) B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control ) or by DEVDase activity measurement ( C ) . Two ( B ) or three ( C ) independent experiments were performed per condition . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) . D - E . Mitochondrial membrane potential ( ΔΨM ) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h ( E ) and quantified in D from two independent experiments . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) . Patient 1 ( P1 ) versus control ( C2 ) : * : p = 0 . 0414 ( 24h ) . Patient 2 ( P2 ) versus control ( C2 ) : * : p = 0 . 0461 ( 24h ) . D - E . Mitochondrial membrane potential ( ΔΨM ) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h ( E ) and quantified in D from two independent experiments . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) . Patient 1 ( P1 ) versus control ( C2 ) : * : p = 0 . 0414 ( 24h ) . Patient 2 ( P2 ) versus control ( C2 ) : * : p = 0 . 0461 ( 24h ) .

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

Figure 7Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001).B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control)B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001).D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).


Figure 8Figure 8 . Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death . Transfections were performed with empty vector ( EV ) or vectors encoding either wild - type CHCHD10 - FLAG ( WT ) or mutant CHCHD10 - FLAG ( P34S and S59L ) . A . Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90 . B - D . HeLa cells transiently expressing the WT or mutated forms of CHCHD10 - FLAG ( P34S or S59L ) were treated with 1 μM Actinomycine D ( ActD ) for 4 , 6 or 8 h with measurement of Annexin V / DAPI staining ( B ) , DEVD - ase activity ( C ) from three independent experiments . Differences between the mutated and non mutated alleles were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) or very significant ( * * : 0 . 01 > p > 0 . 001 ) . P34S versus WT : * * : p = 0 . 0055 ( 4h ) , * : p = 0 . 0103 ( 8h ) . S59L versus WT : * : p = 0 . 0304 ( 4h ) , * : p = 0 . 0129 ( 8h ) . D . Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac . Hsp90 was used as a loading control .

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 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 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

Figure 8Figure 8. Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death. Transfections were performed with empty vector (EV) or vectors encoding either wild-type CHCHD10-FLAG (WT) or mutant CHCHD10-FLAG (P34S and S59L). A. Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90.B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h).D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.


Figure 1Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines : MCF7 parental or doxorubicin - resistant MCF7 / ADR ( ADR , 0 . 5μg / ml ) and irradiation - resistant MCF7 / IR ( IR , 10Gy ) , MDA - MB - 231 parental or doxorubicin - resistant MDA - MB - 231 / ADR ( ADR , 0 . 5μg / ml ) or cisplatin - resistant MDA - MB - 231 / DDP ( 1μg / ml ) , non - small lung cancer cell lines : A549 parental or cisplatin - resistant A549 / DDP ( 1μg / ml ) . MCF7 , MCF7 / ADR and MCF7 / IR , MDA - MB - 231 and MDA - MB - 231 / ADR cells were harvested and mRNA levels of BRD7 were determined by real - time PCR ( n = 3 ) . Values are mean ± SEM and Error bars indicate SEM . Western blot analysis of BRD7 protein levels in MDA - MB - 468 and MDA - MB - 231 cell after treatment with ADR ( 5 μM ) or camptothecin ( CPT ) ( 1 μM ) for different intervals ( n = 3 ) . Representative images of endogenous BRD7 ( green ) and γH2AX foci ( red ) in paraformaldehyde - fixed MDA - MB - 468 cells after treatment with CPT ( 1 μM ) for different intervals . Visualized by immunofluorescence using anti - BRD7 and Alexa Fluor 555 anti - γH2AX antibodies . DNA staining with DAPI ; Scale bars , 2 μm . Quantification of average fluorescence intensity of BRD7 of cells in ( E ) . Error bars indicate SEM ; n > 100 .

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 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 O O O O O O O O O O O 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

Figure 1Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5μg/ml) and irradiation-resistant MCF7/IR (IR, 10Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1μg/ml).MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n=3). Values are mean ± SEM and Error bars indicate SEM.Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n=3).Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm.Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n>100.


Figure 2Silver staining of the BRD7 complex separated by SDS - PAGE . HEK293T cells stably expressing SFB - tagged BRD7 were used for tandem affinity purification ( TAP ) of protein complexes . BRD7 - interacting proteins , including PARP1 and PIK3R2 , are indicated . HEK293T cells transiently transfected with Flag - PARP1 and Myc - BRD7 for 24 h were lysed with RIPA buffer . Followed by immunoprecipitation ( IP ) using antibodies to Myc ( C ) conjugated to agarose followed by Western blot with the indicated antibodies ( n = 3 ) . HEK293T cells transiently transfected with Flag - PARP1 and Myc - BRD7 for 24 h were lysed with RIPA buffer . Followed by immunoprecipitation ( IP ) using antibodies to Flag ( D ) conjugated to agarose followed by Western blot with the indicated antibodies ( n = 3 ) . HeLa and MDA - MB - 231 cells were lysed with RIPA buffer , and lysates were subjected to immunoprecipitation using either anti - IgG , or BRD7 or PARP1 antibodies , and analysed by Western blot ( n = 3 ) . MDA - MB - 231 cells were treated first with Olaparib ( 10 μM ) for 6 h and lysed with RIPA buffer , and lysates were subjected to immunoprecipitation using either anti - IgG or PARP1 antibodies , and analysed by Western blot ( n = 3 ) . Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co - immunoprecipitation using anti - BRD7 or anti - PARP1 antibody . HeLa cell were treated with CPT ( 1 μM , 1 h ) , followed by IP using indicated antibodies , and Western blot was performed . γH2AX was used as a marker of DNA damage induced by CPT ( n = 3 ) .

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 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 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 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 O O O

Figure 2Silver staining of the BRD7 complex separated by SDS-PAGE. HEK293T cells stably expressing SFB-tagged BRD7 were used for tandem affinity purification (TAP) of protein complexes. BRD7-interacting proteins, including PARP1 and PIK3R2, are indicated.HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Myc (C) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to Flag (D) conjugated to agarose followed by Western blot with the indicated antibodies (n=3).HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG, or BRD7 or PARP1 antibodies, and analysed by Western blot (n=3).MDA-MB-231 cells were treated first with Olaparib (10 μM) for 6 h and lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG or PARP1 antibodies, and analysed by Western blot (n=3).Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co-immunoprecipitation using anti-BRD7 or anti-PARP1 antibody. HeLa cell were treated with CPT (1 μM, 1 h), followed by IP using indicated antibodies, and Western blot was performed. γH2AX was used as a marker of DNA damage induced by CPT (n=3).


Figure 3HeLa cells were untreated or treated with CPT ( 1 μM ) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti - IgG or anti - PAR antibodies and immunoblotted with the indicated antibodies ( n = 3 ) . HeLa cells were untreated or treated with CPT ( 1 μM ) for 1 h and cellular lysates were immunoprecipitated using anti - IgG or anti - BRD7 antibodies and immunoblotted were performed using the indicated antibodies ( n = 3 ) . HeLa and 293T cells transfected with Myc - BRD7 plasmid for 24 h were lysed with RIPA buffer . Lysates were then immunoprecipitated using anti - Myc agarose and immunoblotted using the indicated antibodies . Ribosylation levels of exogenous BRD7 were detected using anti - PAR antibody ( n = 3 ) . HeLa cells transfected with Myc - BRD7 plasmid . After 24 hours , cells were treated with either CPT ( 1 μM ) or ADR ( 5 μM ) combined with MG132 ( 10 μM ) for indicated times . Cellular lysates were immunoprecipitated using anti - Myc agarose and immunoblotted using the indicated antibodies ( n = 3 ) . HeLa PARP1 wild - type and PARP1 knockout cells were transfected with Myc - BRD7 for 24 h , lysates were subjected to immunoprecipitation using anti - Myc agarose and analysed by Western blot ( n = 3 ) . HeLa were transfected with BRD7 wild - type and various BRD7 mutant plasmids for 24 h , lysed with RIPA , followed by anti - Myc IP and Western blot with indicated antibody ( n = 3 ) . Ribosylation of BRD7 by PARP1 in vitro . Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin - labeled NAD + . Recombinant proteins were detected by indicated antibodies , and ribosylated proteins were determined with anti - biotin antibody ( n = 3 ) . PAR - binding motif of BRD7 is required for its ribosylation by PARP1 . Recombinant Myc - BRD7 - WT and Myc - BRD7 - mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated ( n = 3 ) .

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 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 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 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 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 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

Figure 3HeLa cells were untreated or treated with CPT (1 μM) for 1 h followed by lysing with RIPA buffer and lysates were then immunoprecipitated using anti-IgG or anti-PAR antibodies and immunoblotted with the indicated antibodies (n=3).HeLa cells were untreated or treated with CPT (1 μM) for 1 h and cellular lysates were immunoprecipitated using anti-IgG or anti-BRD7 antibodies and immunoblotted were performed using the indicated antibodies (n=3).HeLa and 293T cells transfected with Myc-BRD7 plasmid for 24 h were lysed with RIPA buffer. Lysates were then immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies. Ribosylation levels of exogenous BRD7 were detected using anti-PAR antibody (n=3).HeLa cells transfected with Myc-BRD7 plasmid. After 24 hours, cells were treated with either CPT (1 μM) or ADR (5 μM) combined with MG132 (10 μM) for indicated times. Cellular lysates were immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies (n=3).HeLa PARP1 wild-type and PARP1 knockout cells were transfected with Myc-BRD7 for 24 h, lysates were subjected to immunoprecipitation using anti-Myc agarose and analysed by Western blot (n=3).HeLa were transfected with BRD7 wild-type and various BRD7 mutant plasmids for 24 h, lysed with RIPA, followed by anti-Myc IP and Western blot with indicated antibody (n=3).Ribosylation of BRD7 by PARP1 in vitro. Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin-labeled NAD+. Recombinant proteins were detected by indicated antibodies, and ribosylated proteins were determined with anti-biotin antibody (n=3).PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n=3).


Figure 4HeLa cells were transfected with Flag - PARP1 and Myc - BRD7 for 24 h , then treated with MG132 ( 10 μM ) for additional 4 h , and proteins were detected by Western blot with indicated antibodies ( n = 3 ) . HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h , protein levels were detected by Western blot with the indicated antibodies ( n = 3 ) . Expression of BRD7 , p21 and PARP1 measured in PARP1 wild - type and knockout HeLa cells using PARP1 sgRNA by Western blot ( n = 3 ) . MDA - MB - 231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h , cell lysates were analysed by Western blot ( n = 3 ) . MDA - MB - 231 cell were treated with 10 μM of PDD0017273 for indicated times , cell lysates were analysed by Western blot ( n = 3 ) . Wild - type and PARP1 knockout HeLa cells were incubated with 10 μg / ml cycloheximide ( CHX ) for the indicated times . Lysates were harvested and analysed by Western blot . Quantification of BRD7 protein are shown in F ( n = 3 ) and results represent mean ± SEM . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells , respectively . PARP1 wild - type and knockout HeLa cells were transfected with HA - ubiquitin for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h , cells were lysed with RIPA buffer ; followed by anti - BRD7 IP and analysed by Western blot with the indicated antibodies ( n = 3 ) . SFB - BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA - ubiquitin ( Lys48 or Lys63 only ) for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h , lysed with RIPA , subjected to IP using S tag beads followed by Western blot ( n = 3 ) . HeLa cells were co - transfected either wild - type or BRD7 mutant with Flag - PARP1 for 24 h and analysed by Western blot ( n = 3 ) . HeLa cells were co - transfected with either wild - type or BRD7 mutant plus HA - ubiquitin for 24 h , and MG132 ( 10 μM ) was added for an additional 4 h and lysed with RIPA buffer , followed by anti - Myc agarose IP and analysed by Western blot with the indicated antibodies ( n = 3 ) .

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

Figure 4HeLa cells were transfected with Flag-PARP1 and Myc-BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n=3).HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h, protein levels were detected by Western blot with the indicated antibodies (n=3).Expression of BRD7, p21 and PARP1 measured in PARP1 wild-type and knockout HeLa cells using PARP1 sgRNA by Western blot (n=3).MDA-MB-231 cell were treated with different concentration of PARG inhibitor PDD0017273 for 48 h, cell lysates were analysed by Western blot (n=3).MDA-MB-231 cell were treated with 10 μM of PDD0017273 for indicated times, cell lysates were analysed by Western blot (n=3).Wild-type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein are shown in F (n=3) and results represent mean ± SEM. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively.PARP1 wild-type and knockout HeLa cells were transfected with HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h, cells were lysed with RIPA buffer; followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).SFB-BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA-ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n=3).HeLa cells were co-transfected either wild-type or BRD7 mutant with Flag-PARP1 for 24 h and analysed by Western blot (n=3).HeLa cells were co-transfected with either wild-type or BRD7 mutant plus HA-ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti-Myc agarose IP and analysed by Western blot with the indicated antibodies (n=3).


Figure 5HeLa and MDA - MB - 231 cells were transfected with either scrambled or RNF146 siRNAs ( 48 h ) . Protein levels were detected by Western blot . PTEN , which is a known RNF146 - interacting protein , was included as a positive control ( n = 3 ) . HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h , followed by incubation with 10 μg / ml cycloheximide ( CHX ) for the indicated periods of time . Lysates were harvested and analysed by Western blot ( n = 3 ) . Quantification of BRD7 protein levels from B , n = 3 . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h . Control and RNF146 stably overexpressing HeLa cells were transfected with Myc - BRD7 - WT for 24 h , followed by incubation with 10 μg / ml cycloheximide ( CHX ) for indicated periods of time . Lysates were subjected to Western blot analysis ( n = 3 ) . Quantification of BRD7 protein levels from D , n = 3 . Error bars indicate SEM . Relative amounts normalized to the BRD7 protein level at 0 h . HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA - ubiquitin for another 24 h ; MG132 ( 10 μM ) was added for 4 h and lysed with RIPA , followed by anti - BRD7 IP and analysed by Western blot with the indicated antibodies ( n = 3 ) . HeLa cells stably expressing RNF146 were transfected with HA - ubiquitin for 24 h ; MG132 ( 10 μM ) was added for an additional 4 h , followed by anti - BRD7 IP and Western blot ( n = 3 ) .

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 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 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 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

Figure 5HeLa and MDA-MB-231 cells were transfected with either scrambled or RNF146 siRNAs (48 h). Protein levels were detected by Western blot. PTEN, which is a known RNF146-interacting protein, was included as a positive control (n=3).HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analysed by Western blot (n=3). Quantification of BRD7 protein levels from B, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.Control and RNF146 stably overexpressing HeLa cells were transfected with Myc-BRD7-WT for 24 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for indicated periods of time. Lysates were subjected to Western blot analysis (n=3). Quantification of BRD7 protein levels from D, n=3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA-ubiquitin for another 24 h; MG132 (10 μM) was added for 4 h and lysed with RIPA, followed by anti-BRD7 IP and analysed by Western blot with the indicated antibodies (n=3).HeLa cells stably expressing RNF146 were transfected with HA-ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, followed by anti-BRD7 IP and Western blot (n=3).


Figure 6HeLa cells transfected with the indicated plasmids ; after 24 h , cells were lysed with RIPA buffer followed by immunoprecipitation ( IP ) using either anti - Myc or anti - Flag agarose and Western blot with indicated antibody ( n = 3 ) . HeLa and MDA - MB - 231 cells were lysed with RIPA buffer , and lysates were subjected to IP using either anti - IgG , anti - BRD7 or anti - RNF146 antibodies followed by analysis with Western blot ( n = 3 ) . Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co - IP using anti - RNF146 antibody . Cell lysates were incubated with protein - G agarose beads conjugated with indicated antibodies , and Western blot was performed ( n = 3 ) . HeLa cells with wild - type or PARP1 knockout were transfected with Myc - BRD7 - WT for 24 h , their lysates were subjected to IP using anti - Myc agarose and analysed by Western blot with indicated antibodies ( n = 3 ) . HeLa cells stably expressing Flag - RNF146 were transfected with either Myc - BRD7 - WT or Myc - BRD7 - mutant for 24 h , lysates were subjected to IP using anti - Flag agarose and analysed by Western blot with indicated antibodies ( n = 3 ) .

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

Figure 6HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti-Myc or anti-Flag agarose and Western blot with indicated antibody (n=3).HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to IP using either anti-IgG, anti-BRD7 or anti-RNF146 antibodies followed by analysis with Western blot (n=3).Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co-IP using anti-RNF146 antibody. Cell lysates were incubated with protein-G agarose beads conjugated with indicated antibodies, and Western blot was performed (n=3).HeLa cells with wild-type or PARP1 knockout were transfected with Myc-BRD7-WT for 24 h, their lysates were subjected to IP using anti-Myc agarose and analysed by Western blot with indicated antibodies (n=3).HeLa cells stably expressing Flag-RNF146 were transfected with either Myc-BRD7-WT or Myc-BRD7-mutant for 24 h, lysates were subjected to IP using anti-Flag agarose and analysed by Western blot with indicated antibodies (n=3).


Figure 7MDA - MB - 231 cells were transfected twice with indicated siRNAs , the absorbance values of indicated cell lines was measured at different time points by CCK8 assay ( n = 3 ) . Data from three independent experiments are expressed as mean ± SEM . Error bars indicate SEM . Cell lysates treated in A were analysed by western blot with indicated antibodies ( n = 3 ) . MDA - MB - 231 cells were treated with Olaparib ( 10 μM ) combined with ADR ( 0 . 5 μM ) , CPT ( 0 . 5 μM ) or VP16 ( 10 μM ) for 72 h , and CCK8 activity of the cells was detected ( n = 3 ) . Results represent mean ± SEM of three experiments and Error bars indicate SEM . N . S . , not significant ; * * P < 0 . 01 , Student ' s t test . MDA - MB - 231 cells were treated with Olaparib ( 10 μM ) combined with ADR ( 0 . 5 μM ) for 72 h , and their lysates were subjected to IP using anti - BRD7 antibodies and analysed by Western blot with indicated antibodies ( n = 3 ) . MDA - MB - 231 cells were transfected twice with BRD7 siRNAs , and these cells were subjected to CCK8 assay after treatment with Olaparib ( 10 μM ) combined with either ADR ( 0 . 5 μM ) or VP16 ( 10 μM ) for 72 h ( n = 3 ) . Results represent mean ± SEM of three experiments and Error bars indicate SEM . N . S . , not significant ; * * P < 0 . 01 , Student ' s t test . Resistance of BRD7 - depleted cells to Olaparib was reversed by the expression of wild - type BRD7 but not mutant BRD7 . shRNA - resistant BRD7 wild - type and BRD7 mutant plasmids were transduced into BRD7 - depleted MDA - MB - 231 cells , and cell viability was determined as indicated above ( n = 3 ) . Results represent mean ± SEM of three experiments and Error bars indicate SEM . N . S . , not significant ; * * P < 0 . 01 , Student ' s 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 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 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 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 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

Figure 7MDA-MB-231 cells were transfected twice with indicated siRNAs, the absorbance values of indicated cell lines was measured at different time points by CCK8 assay (n=3). Data from three independent experiments are expressed as mean ± SEM. Error bars indicate SEM.Cell lysates treated in A were analysed by western blot with indicated antibodies (n=3).MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM), CPT (0.5 μM) or VP16 (10 μM) for 72 h, and CCK8 activity of the cells was detected (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.MDA-MB-231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM) for 72 h, and their lysates were subjected to IP using anti-BRD7 antibodies and analysed by Western blot with indicated antibodies (n=3).MDA-MB-231 cells were transfected twice with BRD7 siRNAs, and these cells were subjected to CCK8 assay after treatment with Olaparib (10 μM) combined with either ADR (0.5 μM) or VP16 (10 μM) for 72 h (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.Resistance of BRD7-depleted cells to Olaparib was reversed by the expression of wild-type BRD7 but not mutant BRD7. shRNA-resistant BRD7 wild-type and BRD7 mutant plasmids were transduced into BRD7-depleted MDA-MB-231 cells, and cell viability was determined as indicated above (n=3). Results represent mean ± SEM of three experiments and Error bars indicate SEM. N.S., not significant; **P < 0.01, Student's t test.