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 .

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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.


A , 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 .

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A, 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 .

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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 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

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Figure 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 brain


B . 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 .

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B. 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 .

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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 .

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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 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 .

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Figure 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 .

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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 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 .

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Figure 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 ) .

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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 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 .

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Figure 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 O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-C. 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 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 ) .

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Figure 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

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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 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 ) .

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Figure 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 ) .

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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 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 ) .

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Figure 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 )

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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 ) .

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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 ) .

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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 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 .

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Figure 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 ) .

O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.


Western 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 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O

Western 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 .

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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 ) .

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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 .

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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 .

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Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n>100.


Silver 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 .

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Silver 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 ) .

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

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 ) .

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 ) .

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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 ) .

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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 O O O O O 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-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O

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).


HeLa 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 ) .

O O O O O O O B-CONTROLLED_VAR O 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-MEASURED_VAR O O O O O O O O O O O O O

HeLa 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 ) .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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).


HeLa 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 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa 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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 .

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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 ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


HeLa 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 ) .

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

HeLa 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 .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


HeLa 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 ) .

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

HeLa 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 ) .

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 ) .

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

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

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).


MDA - 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 .

MDA-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 ) .

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 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Human red blood cells ( RBCs ) were incubated with 400 nM pigment or control ΔcylE extract , and kinetics of K + and Hb release was monitored . Data shown are the average and SEM of six independent experiments . The time to 50 % K + and 50 % Hb release with pigment was 4 . 8 min and 8 . 4 min , respectively ; n = 6 , P < 0 . 0001 , extra sum - of - squares F test .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human red blood cells (RBCs) were incubated with 400 nM pigment or control ΔcylE extract, and kinetics of K+ and Hb release was monitored. Data shown are the average and SEM of six independent experiments. The time to 50% K+ and 50% Hb release with pigment was 4.8 min and 8.4 min, respectively; n = 6, P < 0.0001, extra sum-of-squares F test.


Role of osmoprotectants in pigment - treated RBC . HumanRBCs were pre - incubated with GBSpigment for 2 min at RT , centrifuged , and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0 . 40 nm ( PEG200 ) , 0 . 56 nm ( PEG400 ) , 0 . 89 nm ( PEG1000 ) , 1 . 1 nm ( PEG1500 ) , or 1 . 6 nm ( PEG3000 ) , respectively . Release of Hb was measured after 1 h of incubation at 37 ° C . Data shown are the average and SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

Role of osmoprotectants in pigment-treated RBC. HumanRBCs were pre-incubated with GBSpigment for 2 min at RT, centrifuged, and resuspended in the presence and absence of 30 mM osmoprotectant with hydrodynamic radius of 0.40 nm (PEG200), 0.56 nm (PEG400), 0.89 nm (PEG1000), 1.1 nm (PEG1500), or 1.6 nm (PEG3000), respectively. Release of Hb was measured after 1 h of incubation at 37°C. Data shown are the average and SEM of three independent experiments.


Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers . Lipid bilayers were generated using diphytanoylphosphatidylcholine ( DPhPC ) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract . In the pigment - treated sample , channel conductance indicating disruption of the membrane is seen within 45 s . Erratic and non - discrete fluctuations in current are observed , suggesting the formation of multiple , small membrane defects . The bilayer eventually breaks at 120 s . In lipid bilayers treated with the control ΔcylE extract , the mean ionic current trace remains constant at 0 pA , showing no membrane disruption . Data shown are representative of three independent experiments .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Characteristics of membrane permeabilization by the GBSpigment in artificial lipid bilayers. Lipid bilayers were generated using diphytanoylphosphatidylcholine (DPhPC) and treated with either 2 μM pigment or an equivalent amount of the control ΔcylE extract. In the pigment-treated sample, channel conductance indicating disruption of the membrane is seen within 45 s. Erratic and non-discrete fluctuations in current are observed, suggesting the formation of multiple, small membrane defects. The bilayer eventually breaks at 120 s. In lipid bilayers treated with the control ΔcylE extract, the mean ionic current trace remains constant at 0 pA, showing no membrane disruption. Data shown are representative of three independent experiments.


A , B PBMC - derived macrophages were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by ELISA ( B ) . Data information : Data shown are the average of four independent experiments performed in triplicate , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA . ( A ) n = 4 , * P = 0 . 021 for WT versus ΔcovR ; * P = 0 . 01 for ΔcovR versus ΔcovRΔcylE . ( B ) n = 4 , * P = 0 . 031 for WT versus ΔcovR ; * P = 0 . 036 for ΔcovR versus ΔcovRΔcylE . ( C ) n = 4 , * * * * P < 0 . 0001 , * * P = 0 . 002 . ( D ) n = 4 , * * P = 0 . 005 .

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

A, B PBMC-derived macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by ELISA (B).Data information: Data shown are the average of four independent experiments performed in triplicate, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 4, *P = 0.021 for WT versus ΔcovR; *P = 0.01 for ΔcovR versus ΔcovRΔcylE. (B) n = 4, *P = 0.031 for WT versus ΔcovR; *P = 0.036 for ΔcovR versus ΔcovRΔcylE. (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.


C , D PBMC - derived macrophages primed with 100 ng / ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 4 , * * * * P < 0 . 0001 , * * P = 0 . 002 . ( D ) n = 4 , * * P = 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D PBMC-derived macrophages primed with 100 ng/ml LPS for 3 h were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D). (C) n = 4, ****P < 0.0001, **P = 0.002. (D) n = 4, **P = 0.005.


A , B WT THP - 1macrophages were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data information : Data from three independent experiments performed in triplicate are shown , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA . ( A ) n = 3 , * * P = 0 . 0080 for WT versus ΔcovR ; * * P = 0 . 0016 for ΔcovR versus ΔcovRΔcylE . ( B ) n = 3 , * P = 0 . 03 for WT versus ΔcovR ; * P = 0 . 02 for ΔcovR versus ΔcovRΔcylE . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * P = 0 . 01 .

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

A, B WT THP-1macrophages were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B).Data information: Data from three independent experiments performed in triplicate are shown, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA. (A) n = 3, **P = 0.0080 for WT versus ΔcovR; **P = 0.0016 for ΔcovR versus ΔcovRΔcylE. (B) n = 3, *P = 0.03 for WT versus ΔcovR; *P = 0.02 for ΔcovR versus ΔcovRΔcylE. (C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.


C , D WT THP - 1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release from pigment - or ΔcylE extract - treated cells was measured by Luminex assay ( D ) . ( C ) n = 3 , * * * * P < 0 . 0001 . ( D ) n = 3 , * P = 0 . 01 .

O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D WT THP-1macrophages were incubated with various concentrations of GBSpigment or control ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release from pigment- or ΔcylE extract-treated cells was measured by Luminex assay (D).(C) n = 3, ****P < 0.0001. (D) n = 3, *P = 0.01.


A , B THP - 1macrophages transfected with empty vector , scrambled control , shASC , or shNLRP3 were treated with GBS WT , ΔcylE , ΔcovR , or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h . Cytotoxicity was measured by LDH release ( A ) , and IL1β release in supernatants was measured by Luminex assay ( B ) . Data from three independent experiments performed in triplicate are shown . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( A ) n = 3 , for WT : * * P = 0 . 001 ( vector versus shNLRP3 ) , * * P = 0 . 0016 ( vector versus shASC ) ; for ΔcovR : * * * P = 0 . 0008 ( vector versus shNLRP3 ) , * * * P = 0 . 0001 ( vector versus shASC ) , * P = 0 . 047 ( scrambled versus shNLRP3 ) , * * P = 0 . 0054 ( scrambled versus shASC ) . Data obtained from THP - 1 / vector was not significantly different from THP - 1 / scrambled , P = 0 . 18 . ( B ) n = 3 , * P = 0 . 025 . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 89 .

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

A, B THP-1macrophages transfected with empty vector, scrambled control, shASC, or shNLRP3 were treated with GBS WT, ΔcylE, ΔcovR, or ΔcovRΔcylE at an MOI of 1 and incubated for 4 h. Cytotoxicity was measured by LDH release (A), and IL1β release in supernatants was measured by Luminex assay (B). Data from three independent experiments performed in triplicate are shown.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (A) n = 3, for WT: **P = 0.001 (vector versus shNLRP3), **P = 0.0016 (vector versus shASC); for ΔcovR: ***P = 0.0008 (vector versus shNLRP3), ***P = 0.0001 (vector versus shASC), *P = 0.047 (scrambled versus shNLRP3), **P = 0.0054 (scrambled versus shASC). Data obtained from THP-1/vector was not significantly different from THP-1/scrambled, P = 0.18. (B) n = 3, *P = 0.025. Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.89.


C , D The shRNA THP - 1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h . Cytotoxicity was measured by alamar blue assay ( C ) , and IL1β release in supernatants was measured by Luminex assay ( D ) . Pigment - mediated cytotoxicity is dependent on the NLRP3 inflammasome components , suggesting that pigment is inducing a programmed cell death . Data shown are the average of at least three independent experiments performed in triplicate . Data information : Data were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM . ( C ) n = 3 , for 4 μM pigment : * * P = 0 . 0063 ( vector versus shNLRP3 ) , * * P = 0 . 0035 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 ) , * * P = 0 . 009 ( scrambled versus shASC ) ; for 2 μM pigment : * * P = 0 . 0088 ( vector versus shNLRP3 ) , * * P = 0 . 0066 ( vector versus shASC ) , * P = 0 . 01 ( scrambled versus shNLRP3 or shASC ) ; for 1 μM pigment : * P = 0 . 02 ( vector versus shNLRP3 ) , * P = 0 . 01 ( vector versus shASC ) . Data obtained from THP - 1 / vector were not significantly different from THP - 1 / scrambled , P = 0 . 99 . ( D ) n = 3 , * * * * P < 0 . 0001 , * P = 0 . 015 .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D The shRNA THP-1macrophages were incubated with various concentrations of GBSpigment or ΔcylE extract for 4 h. Cytotoxicity was measured by alamar blue assay (C), and IL1β release in supernatants was measured by Luminex assay (D). Pigment-mediated cytotoxicity is dependent on the NLRP3 inflammasome components, suggesting that pigment is inducing a programmed cell death. Data shown are the average of at least three independent experiments performed in triplicate.Data information: Data were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM. (C) n = 3, for 4 μM pigment: **P = 0.0063 (vector versus shNLRP3), **P = 0.0035 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3), **P = 0.009 (scrambled versus shASC); for 2 μM pigment: **P = 0.0088 (vector versus shNLRP3), **P = 0.0066 (vector versus shASC), *P = 0.01 (scrambled versus shNLRP3 or shASC); for 1 μM pigment: *P = 0.02 (vector versus shNLRP3), *P = 0.01 (vector versus shASC). Data obtained from THP-1/vector were not significantly different from THP-1/scrambled, P = 0.99. (D) n = 3, ****P < 0.0001, *P = 0.015.


A , B THP - 1macrophages proficient for NLRP3 ( transfected with scrambled control , A ) or deficient for NLRP3 ( shNLRP3 , B ) were treated with 1 μM pigment or ΔcylE extract for 20 min , and propidium iodide ( PI ) was added during the final 10 min . PI uptake was measured by flow cytometry , and data shown are representative of two independent experiments .

O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B THP-1macrophages proficient for NLRP3 (transfected with scrambled control, A) or deficient for NLRP3 (shNLRP3, B) were treated with 1 μM pigment or ΔcylE extract for 20 min, and propidium iodide (PI) was added during the final 10 min. PI uptake was measured by flow cytometry, and data shown are representative of two independent experiments.


C , D Intracellular potassium concentration was measured by ICP - AES . THP - 1macrophages transfected with the scrambled control ( C ) or shNLRP3 ( D ) were treated with GBSpigment ( 1 μM ) or an equivalent amount of the ΔcylE extract . At various time points , cells were lysed and intracellular [ K + ] was measured relative to untreated cells ( see bars and left y - axis ) , and percent cell death was quantified by alamar blue ( see squares , dotted connecting lines and right y - axis ) . Both NLRP3 - proficient and NLRP3 - deficient macrophages initially lose intracellular K + due to GBSpigment ( compare t = 0 min to t = 30 min ) , but the NLRP3 - deficient cells ( shNLRP3 ) are able to recover , while the scrambled control do not , demonstrating that initial K + loss occurs independently of NLRP3 . Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett ' s multiple comparison test following ANOVA ; all data were compared to control at t = 0 , error bars ± SEM . ( C ) n = 3 , * * * P = 0 . 0002 , * P = 0 . 019 , * * P = 0 . 0032 for 120 min , * * P = 0 . 0072 for 180 min . ( D ) n = 3 , * * P = 0 . 0043 , * P = 0 . 031 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Intracellular potassium concentration was measured by ICP-AES. THP-1macrophages transfected with the scrambled control (C) or shNLRP3 (D) were treated with GBSpigment (1 μM) or an equivalent amount of the ΔcylE extract. At various time points, cells were lysed and intracellular [K+] was measured relative to untreated cells (see bars and left y-axis), and percent cell death was quantified by alamar blue (see squares, dotted connecting lines and right y-axis). Both NLRP3-proficient and NLRP3-deficient macrophages initially lose intracellular K+ due to GBSpigment (compare t = 0 min to t = 30 min), but the NLRP3-deficient cells (shNLRP3) are able to recover, while the scrambled control do not, demonstrating that initial K+ loss occurs independently of NLRP3. Data are average of three independent experiments performed with independent pigment preparations in triplicate and were analyzed using Dunnett's multiple comparison test following ANOVA; all data were compared to control at t = 0, error bars ± SEM. (C) n = 3, ***P = 0.0002, *P = 0.019, **P = 0.0032 for 120 min, **P = 0.0072 for 180 min. (D) n = 3, **P = 0.0043, *P = 0.031.


( E ) WT THP - 1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride , and cytotoxicity was measured by alamar blue assay . The addition of potassium chloride is able to protect the macrophages from cytolysis , demonstrating that K + efflux is essential for this process . Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni ' s multiple comparison test following ANOVA , error bars ± SEM ( n = 3 , * * * * P < 0 . 0001 , * * P = 0 . 0036 ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) WT THP-1macrophages were incubated with pigment in media containing either 5 mM or 50 mM potassium chloride, and cytotoxicity was measured by alamar blue assay. The addition of potassium chloride is able to protect the macrophages from cytolysis, demonstrating that K+ efflux is essential for this process. Data shown are three independent experiments performed in triplicate and were analyzed using Bonferroni's multiple comparison test following ANOVA, error bars ± SEM (n = 3, ****P < 0.0001, **P = 0.0036).


THP - 1macrophages were treated with GBSpigment , and caspase 1 activation was measured by flow cytometry using a FLICA reagent . Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line , demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome . Results are representative of three independent experiments .

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

THP-1macrophages were treated with GBSpigment, and caspase 1 activation was measured by flow cytometry using a FLICA reagent. Pigment treatment of the scrambled shRNA control cell line induces more caspase 1 activation compared to the shNLRP3cell line, demonstrating that the pigment activates caspase 1 exclusively through the NLRP3 inflammasome. Results are representative of three independent experiments.


WT THP - 1macrophages were treated with the caspase 1 inhibitor Z - YVAD - FMK , the caspase 3 / 7 inhibitor Z - DEVD - FMK , or DMSO only prior to treatment with the GBSpigment . YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment , demonstrating that caspase 1 is required for GBSpigment - mediated cell death , characteristic of pyroptosis , while DEVD had no effect . Data are average of three independent experiments , error bars ± SEM . Significance was determined using Bonferroni ' s multiple comparison test following ANOVA ( n = 3 , * * * * P < 0 . 0001 , * * * P = 0 . 0002 , * P = 0 . 014 ) .

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT THP-1macrophages were treated with the caspase 1 inhibitor Z-YVAD-FMK, the caspase 3/7 inhibitor Z-DEVD-FMK, or DMSO only prior to treatment with the GBSpigment. YVAD is able to significantly decrease cytotoxicity in cells treated with GBSpigment, demonstrating that caspase 1 is required for GBSpigment-mediated cell death, characteristic of pyroptosis, while DEVD had no effect. Data are average of three independent experiments, error bars ± SEM. Significance was determined using Bonferroni's multiple comparison test following ANOVA (n = 3, ****P < 0.0001, ***P = 0.0002, *P = 0.014).


In utero fetal death in wild - type CD - 1mice due to infection with GBS WT , hyperhemolytic ΔcovR , and non - hemolytic ΔcovRΔcylE . Fetal death is represented by the number of dead fetuses / total number of fetuses obtained from six pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * * * * P < 0 . 0001 , Fisher ' s exact test .

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

In utero fetal death in wild-type CD-1mice due to infection with GBS WT, hyperhemolytic ΔcovR, and non-hemolytic ΔcovRΔcylE. Fetal death is represented by the number of dead fetuses/total number of fetuses obtained from six pregnant mice per group. 'n' indicates total number of pups (both live and dead); ****P < 0.0001, Fisher's exact test.


H & amp ; amp ; E staining of uterine tissue . Open arrow indicates the presence of few mononuclear cells , whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris .

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H&amp;E staining of uterine tissue. Open arrow indicates the presence of few mononuclear cells, whereas filled arrows indicate increased infiltration of inflammatory cells and necrotic debris.


Fetal death due to infection with hyperhemolytic GBS ΔcovR and non - hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome - deficient mice ; fetal death is represented by the number of dead fetuses / total number of fetuses obtained from 6 pregnant mice per group . ' n ' indicates total number of pups ( both live and dead ) ; * P = 0 . 011 , * * P = 0 . 0015 , * * * * P < 0 . 0001 , Fisher ' s exact test . Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS ; P = 0 . 31 , Fisher ' s exact test .

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

Fetal death due to infection with hyperhemolytic GBS ΔcovR and non-hemolytic ΔcovRΔcylE in WT C57BL6 and NLRP3 inflammasome-deficient mice; fetal death is represented by the number of dead fetuses/total number of fetuses obtained from 6 pregnant mice per group. 'n' indicates total number of pups (both live and dead); *P = 0.011, **P = 0.0015, ****P < 0.0001, Fisher's exact test. Fetal death due to ΔcovRΔcylE in WT C57BL6 and NLRP3KO mice was not significant and is indicated as NS; P = 0.31, Fisher's exact test.


Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains ( n = 6 / pup ; of note , pups that were delivered preterm were excluded from CFU enumeration . Scheme of pup numbering is shown in ( A ) . RUH and LUH indicate right uterine horn and left uterine horn , respectively . CFUs are not significantly different between any of the groups ( ANOVA , P = 0 . 6 , error bars ± SEM ) .

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

Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.Bacterial burden in fetal pups and uterine horns from mice infected with the various GBS strains (n = 6/pup; of note, pups that were delivered preterm were excluded from CFU enumeration. Scheme of pup numbering is shown in (A). RUH and LUH indicate right uterine horn and left uterine horn, respectively. CFUs are not significantly different between any of the groups (ANOVA, P = 0.6, error bars ± SEM).


Female pregnant wild - type ( CD - 1 , C57BL6 ) or NLRP3 - deficient mice were injected in utero with 106 - 7CFU of GBS WT , ΔcovR , or ΔcovRΔcylE and monitored for preterm birth . Surgery and GBS inoculation for each pregnant mouse were performed independently . Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls . IL - 1β levels in GBS - infected tissues ( placenta and fetus , n = 6 / group ) was measured by Luminex assay ( * P = 0 . 025 , * * * P = 0 . 0002 , * * * * P < 0 . 0001 , Bonferroni ' s multiple comparison test following ANOVA ) . IL - 1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS ; P = 0 . 99 .

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

Female pregnant wild-type (CD-1, C57BL6) or NLRP3-deficient mice were injected in utero with 106-7CFU of GBS WT, ΔcovR, or ΔcovRΔcylE and monitored for preterm birth. Surgery and GBS inoculation for each pregnant mouse were performed independently. Data shown are representative of experiments with 6 animals per group for each GBS strain and two animals were used for saline controls.IL-1β levels in GBS-infected tissues (placenta and fetus, n = 6/group) was measured by Luminex assay (*P = 0.025, ***P = 0.0002, ****P < 0.0001, Bonferroni's multiple comparison test following ANOVA). IL-1β levels was not significantly different in NLRP3KO mice infected with ΔcovR compared to NLRP3KO mice infected with ΔcovRΔcylEmice and is indicated as NS; P = 0.99.


( d ) NIH3T3 cells stably expressing PS - CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels .

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(d) NIH3T3 cells stably expressing PS-CFP2 proteins were exposed to a 405 nm light for the indicated times and photoconverting was monitored by imaging cells in the green and blue channels.


( e , f ) Mouse fibroblasts ( NIH3T3 ) stably expressing PS - CFP2 or KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . ( e ) Representative images in both channels . ( f ) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( white bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of four different experiments with > 50 cells counted per experiment . ( * ) Differences with PS - CFP2 in the green channel ( P = 0 . 005 , ANOVA - Bonferroni ) . ( § ) Differences with samples in the presence of serum ( P = 0 . 03 , ANOVA - Bonferroni ) . ( g ) Green fluorescence intensity at excitation 450 nm and emission 510 - 550 nm . Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence ( white bars ) or absence ( gray bars ) of serum was calculated for each condition . Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means + s . e . of 3 independent experiments . Differences with serum ( * ) were significant for P = 0 . 04 ( t - test ) . Scale bars , 5 μm .

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

(e, f) Mouse fibroblasts (NIH3T3) stably expressing PS-CFP2 or KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. (e) Representative images in both channels. (f) Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (white bars) or absence (gray bars) of serum. Values are mean+s.e. of four different experiments with >50 cells counted per experiment. (*) Differences with PS-CFP2 in the green channel (P=0.005, ANOVA-Bonferroni). (§) Differences with samples in the presence of serum (P=0.03, ANOVA-Bonferroni). (g) Green fluorescence intensity at excitation 450 nm and emission 510-550 nm. Fluorescence intensity in each sample was normalized by the number of cells present in the sample and the percentage of intensity at time 0 still remaining after 16 h of incubation in the presence (white bars) or absence (gray bars) of serum was calculated for each condition. Values are expressed relative to the decay observed in cells maintained in the presence of serum and are means+s.e. of 3 independent experiments. Differences with serum (*) were significant for P=0.04 (t-test). Scale bars, 5 μm.


( a - c ) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ - PS - CFP2 maintained in the absence ( black circles ) or presence ( white squares ) of serum for the indicated times . ( a ) Representative images of cells maintained in serum - free media . Quantification of the number of puncta per cell ( b ) and green fluorescence intensity ( c ) . Values are expressed as percentage of fluorescence at time 0 and half - lives ( t1 / 2 ) are indicated . Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum ( * ) were significant for P = 0 . 048 ( 16 h ) and P = 0 . 04 ( 24 h ) ( t - test ) .

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(a-c) Temporal changes in the green fluorescence pattern of photoactivated NIH3T3 cells stably expressing KFERQ-PS-CFP2 maintained in the absence (black circles) or presence (white squares) of serum for the indicated times. (a) Representative images of cells maintained in serum-free media. Quantification of the number of puncta per cell (b) and green fluorescence intensity (c). Values are expressed as percentage of fluorescence at time 0 and half-lives (t1/2) are indicated. Differences in the number of puncta and fluorescence intensity between cells maintained in the presence and absence of serum (*) were significant for P=0.048 (16 h) and P=0.04 (24 h) (t-test).


( d ) The same cells imaged in the cyan channel , before and at the indicated times after photoconversion ( arrow ) by exposure to 405 nm light . Top : Representative images of the cyan fluorescence . Bottom : Quantification of the intensity of the cyan fluorescence . Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1 . Values are mean + s . e . of 3 different experiments with > 10 cells counted per experiment . Scale bars , 5 μm .

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(d) The same cells imaged in the cyan channel, before and at the indicated times after photoconversion (arrow) by exposure to 405 nm light. Top: Representative images of the cyan fluorescence. Bottom: Quantification of the intensity of the cyan fluorescence. Values are expressed as times the intensity before photoconversion that was given an arbitrary value of 1. Values are mean+s.e. of 3 different experiments with >10 cells counted per experiment. Scale bars, 5 μm.


( a ) Localization of photoconverted KFERQ - PS - CFP2 ( green ) in LysoTracker - stained compartments ( red ) in mouse fibroblasts maintained in the presence or absence of serum .

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(a) Localization of photoconverted KFERQ-PS-CFP2 (green) in LysoTracker-stained compartments (red) in mouse fibroblasts maintained in the presence or absence of serum.


( b ) Percentage of co - localization of KFERQ - PS - CFP2 with the indicated proteins . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment .

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(b) Percentage of co-localization of KFERQ-PS-CFP2 with the indicated proteins. Values are mean+s.e. of three different experiments with >50 cells counted per experiment.


( c ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LAMP - 1 , LAMP - 2 or LAMP - 2A .

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(c) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LAMP-1, LAMP-2 or LAMP-2A.


( d ) Immunofluorescence with antibodies that recognize PS - CFP2 ( green ) and hsc70 ( red ) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction .

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(d) Immunofluorescence with antibodies that recognize PS-CFP2 (green) and hsc70 (red) in cells maintained in the absence of serum and fixed with methanol to eliminate the soluble cytosolic fraction.


( e , f ) Co - localization of photoconverted KFERQ - PS - CFP2 ( green ) with LC3 , CD - M6PR , Rab5 ( e ) and ubiquitin ( f ) in mouse fibroblasts maintained in serum - free media . Scale bars , 5 μm .

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(e, f) Co-localization of photoconverted KFERQ-PS-CFP2 (green) with LC3, CD-M6PR, Rab5 (e) and ubiquitin (f) in mouse fibroblasts maintained in serum-free media. Scale bars, 5 μm.


( a ) Mouse fibroblasts knockeddown for LAMP - 2A and transfected with KFERQ - PS - CFP2 were photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Representative images of cells also immunostained for LAMP - 1 are shown .

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(a) Mouse fibroblasts knockeddown for LAMP-2A and transfected with KFERQ-PS-CFP2 were photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Representative images of cells also immunostained for LAMP-1 are shown.


( b , c ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were photoconverted and maintained for 16 h in media supplemented ( + ) or not ( − ) with serum in the presence of 3 - methyladenine ( 3 - MA ) to inhibit macroautophagy . ( b ) Representative images of the 3 - MA - treated cells . ( c ) Quantification of the number of green fluorescence puncta per cell in 3 - MA treated or not cells maintained in the presence ( white bars ) or absence ( black bars ) of serum . Values are mean + s . e . of 3 different experiments with > 50 cells counted per experiment . No significant differences were detectable between samples supplemented or not with 3MA . Scale bars , 5 μm .

O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b, c) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were photoconverted and maintained for 16 h in media supplemented (+) or not (−) with serum in the presence of 3-methyladenine (3-MA) to inhibit macroautophagy. (b) Representative images of the 3-MA-treated cells. (c) Quantification of the number of green fluorescence puncta per cell in 3-MA treated or not cells maintained in the presence (white bars) or absence (black bars) of serum. Values are mean+s.e. of 3 different experiments with >50 cells counted per experiment. No significant differences were detectable between samples supplemented or not with 3MA. Scale bars, 5 μm.


( a ) Immunoblot with the antibody that recognizes PS - CFP2 of homogenates ( Hom ) , cytosol ( Cyt ) and lysosomal membranes ( MB ) and matrices ( Mtx ) isolated from mouse fibroblasts stably expressing PS - CFP2 or KFERQ - PS - CFP2 proteins 16 h after serum removal .

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(a) Immunoblot with the antibody that recognizes PS-CFP2 of homogenates (Hom), cytosol (Cyt) and lysosomal membranes (MB) and matrices (Mtx) isolated from mouse fibroblasts stably expressing PS-CFP2 or KFERQ-PS-CFP2 proteins 16 h after serum removal.


( b ) Immunoblot for the indicated proteins of homogenates ( Hom ) , mitochondria ( Mit ) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ - PS - CFP2 maintained for 16 h in media supplemented ( + ) or not ( − ) with serum . ( c ) Quantification of the amount of KFERQ - PS - CFP2 associated to lysosomes ( matrix and membrane ) performed in blots as the one shown in ( b ) . Values are expressed as percentage of the total KFERQ - PS - CFP2 in the cell and are mean + s . e . of three different experiments . Difference in the lysosomal associated KFERQ - PS - CFP2 between serum + and serum − condition ( * ) were significant for P = 0 . 033 ( t - test ) .

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(b) Immunoblot for the indicated proteins of homogenates (Hom), mitochondria (Mit) and lysosomal membranes and matrices isolated from cells stably expressing KFERQ-PS-CFP2 maintained for 16 h in media supplemented (+) or not (−) with serum. (c) Quantification of the amount of KFERQ-PS-CFP2 associated to lysosomes (matrix and membrane) performed in blots as the one shown in (b). Values are expressed as percentage of the total KFERQ-PS-CFP2 in the cell and are mean+s.e. of three different experiments. Difference in the lysosomal associated KFERQ-PS-CFP2 between serum+ and serum− condition (*) were significant for P=0.033 (t-test).