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 ) .

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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 ) .

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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 ) .

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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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O 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 O O O O O O O O O B-MEASURED_VAR 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 ) .

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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 ) .

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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 ) .

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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 ) .

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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 ) .

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

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 ) .

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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 ) .

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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 ) .

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

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 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 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 B-MEASURED_VAR 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

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 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 ) .

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

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 B-MEASURED_VAR O B-MEASURED_VAR 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

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 ) .

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

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 B-CONTROLLED_VAR 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

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 .

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

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 B-CONTROLLED_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 O B-CONTROLLED_VAR 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 ) .

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

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

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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 O O O O O O O O O O O O O O O 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-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 .

(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 .

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(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).


( d ) Changes in the intracellular content of KFERQ - PS - CFP2 on serum removal . Values are expressed as percentage of KFERQ - PS - CFP2 present in serum - supplemented cells and area mean + s . e . of three different experiments . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 007 ( t - test ) .

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(d) Changes in the intracellular content of KFERQ-PS-CFP2 on serum removal. Values are expressed as percentage of KFERQ-PS-CFP2 present in serum-supplemented cells and area mean+s.e. of three different experiments. Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.007 (t-test).


( e - g ) Effect of inhibition of macroautophagy by treatment with 3 - methyladenine ( 3 - MA ) on the lysosomal content of KFERQ - PS - CFP2 in cells maintained in serum - free media for 16 h . ( e ) Immunoblot for the indicated proteins of the same fractions as described in ( b ) . The amount of KFERQ - PS - CFP2 associated to lysosomes ( f ) and the changes in the intracellular content of KFERQ - PS - CFP2 were calculated as in ( c , d ) . Values are mean + s . e . of three different experiments and differences are significant ( * ) for P = 0 . 032 ( t - test ) .

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(e-g) Effect of inhibition of macroautophagy by treatment with 3-methyladenine (3-MA) on the lysosomal content of KFERQ-PS-CFP2 in cells maintained in serum-free media for 16 h. (e) Immunoblot for the indicated proteins of the same fractions as described in (b). The amount of KFERQ-PS-CFP2 associated to lysosomes (f) and the changes in the intracellular content of KFERQ-PS-CFP2 were calculated as in (c, d). Values are mean+s.e. of three different experiments and differences are significant (*) for P=0.032 (t-test).


( a , b ) Human neuroblastoma ( SH - SY5Y ) , hepatoma ( HuH - 7 ) and breast adenocarcinoma ( MCF - 7 ) cell lines were transiently transfected with KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Images show representative cells after immunostaining for LAMP - 2 ( red ) . ( b ) Quantification of the number of green fluorescent puncta per cell under basal conditions ( black bars ) and after serum removal ( gray bars ) . Values of NIH3T3 are included as reference . Values are mean + s . e . of three - to - five different experiments with > 50 cells counted per experiment . Differences with ( * ) NIH3T3 or ( § ) with cell supplemented with serum are significant for P0 . 05 ( range 0 . 002 - 0 . 03 , ANOVA - Bonferroni ) .

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(a, b) Human neuroblastoma (SH-SY5Y), hepatoma (HuH-7) and breast adenocarcinoma (MCF-7) cell lines were transiently transfected with KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Images show representative cells after immunostaining for LAMP-2 (red). (b) Quantification of the number of green fluorescent puncta per cell under basal conditions (black bars) and after serum removal (gray bars). Values of NIH3T3 are included as reference. Values are mean+s.e. of three-to-five different experiments with >50 cells counted per experiment. Differences with (*) NIH3T3 or (§) with cell supplemented with serum are significant for P0.05 (range 0.002-0.03, ANOVA-Bonferroni).


( c ) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ - PS - CFP2 , photoconverted and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : representative image in the green channel . Right : Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence ( black bars ) or absence ( gray bars ) of serum . Values are mean + s . e . of three different experiments with > 50 cells counted per experiment . ( P = 0 . 002 , ANOVA - Bonferroni ) .

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(c) Primary mouse skin fibroblasts were transduced with a lentiviral vector for expression of KFERQ-PS-CFP2, photoconverted and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: representative image in the green channel. Right: Quantification of the number of green fluorescent puncta per cell in cells maintained in the presence (black bars) or absence (gray bars) of serum. Values are mean+s.e. of three different experiments with >50 cells counted per experiment. (P=0.002, ANOVA-Bonferroni).


( d ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence ( white bar ) or absence ( black bars ) of serum and subjected to treatment with 3 - methyladenine ( 3 - MA ) , the proteasome inhibitor MG - 132 or H2O2 . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . Scale bars , 5 μm .

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

(d) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence (white bar) or absence (black bars) of serum and subjected to treatment with 3-methyladenine (3-MA), the proteasome inhibitor MG-132 or H2O2. In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.


( e ) Mouse fibroblasts stably expressing KFERQ - PS - CFP2 were plated in 384 - well plates , maintained in the presence of serum supplemented media and leave untreated ( white bar ) or subjected to the indicated treatments ( black bars ) . In both ( d , e ) , after fixation , images were subjected to high content image analysis and the percent of responders was calculated in > 200 cells per condition . Values are mean + s . e . of results in three different experiments . * P0 . 001 ( range 0 . 0002 - 0 . 0007 , t - test ) . Scale bars , 5 μm .

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(e) Mouse fibroblasts stably expressing KFERQ-PS-CFP2 were plated in 384-well plates, maintained in the presence of serum supplemented media and leave untreated (white bar) or subjected to the indicated treatments (black bars). In both (d, e), after fixation, images were subjected to high content image analysis and the percent of responders was calculated in >200 cells per condition. Values are mean+s.e. of results in three different experiments. *P0.001 (range 0.0002-0.0007, t-test). Scale bars, 5 μm.


( b ) Mouse fibroblasts ( NIH3T3 ) stably expressing KFERQ - PA - mCherry1 were photoactivated and maintained in media supplemented ( + ) or not ( − ) with serum for 16 h . Left : Representative images . Nuclei are staining with DAPI . Right : Quantification of the number of red fluorescent puncta per cell . Values are mean + s . e . of three different experiments with > 20 cells counted per experiment . * P = 0 . 001 , ANOVA - Bonferroni .

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(b) Mouse fibroblasts (NIH3T3) stably expressing KFERQ-PA-mCherry1 were photoactivated and maintained in media supplemented (+) or not (−) with serum for 16 h. Left: Representative images. Nuclei are staining with DAPI. Right: Quantification of the number of red fluorescent puncta per cell. Values are mean+s.e. of three different experiments with >20 cells counted per experiment. *P=0.001, ANOVA-Bonferroni.


( c ) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ - PA - mCherry1 at the indicated times after photoactivation using FACS analysis . Cells were maintained in media supplemented ( white squares ) or not ( black circles ) with serum , and fluorescence by the MFI was calculated on events gated on living cells . Values are expressed as percentage of the intensity at time 0 and are mean + s . e . of 3 - 4 individual experiments .

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(c) Temporal changes in the mCherry fluorescent intensity in cells stably expressing KFERQ-PA-mCherry1 at the indicated times after photoactivation using FACS analysis. Cells were maintained in media supplemented (white squares) or not (black circles) with serum, and fluorescence by the MFI was calculated on events gated on living cells. Values are expressed as percentage of the intensity at time 0 and are mean+s.e. of 3-4 individual experiments.


( d ) Representative FACS experiment showing the effect of serum removal at 6 h ( top ) and 42 h ( bottom ) . PS , photoswitching . Scale bars , 5 μm .

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(d) Representative FACS experiment showing the effect of serum removal at 6 h (top) and 42 h (bottom). PS, photoswitching. Scale bars, 5 μm.


Mouse qPCR array analysis was performed with Colo357 and MiaPaca - 2 orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect changes of mouse stromal gene expression after treatment . A heat map was generated with ratios of gene expression ( 2G8 vs . control ) .

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Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control).


ELISA - based Quansys mouse chemokine assay was performed with Capan - 1 ( Cap ) , MiaPaCa - 2 ( Mia ) , Colo357 ( Colo ) , and C5LM2 ( C5 ) orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect mouse chemokine changes after treatment . Change of mouse IL - 6 was shown .

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ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown.


KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Tumors from were collected for mouse IL - 6 ELISA ( n = 6 / group ) .

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KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group).


KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . The activation of SMAD2 ( P - Ser465 / 467 ) ( E and H - I ) , STAT3 ( P - Tyr705 ) ( F and J - K ) and expression of IL - 6R ( G ) was detected by immunohistochemistry ( n = 4 / group ) .

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KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).


Single - cell RNA sequencing was performed to profile cell populations in normal mouse pancreas ( n = 2 ) , early KIC ( 40 - day - old , n = 2 ) and late KIC ( 60 - day - old , n = 3 ) pancreata . Samples from the same stage were pooled . Violin plots of expression of Il6 , Tgfbr1 and Tgfbr2 in distinct cell populations is shown .

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Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown.


Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC ( mPLRB8 , mPLRB9 ) , KPC ( KPC - M01 , KPC - M09 ) and KPfC ( BMFA3 , CT1BA5 ) mouse cancer cells , mouse macrophages ( RAW 264 . 7 ) , and mouse fibroblasts ( NIH 3T3 and pancreatic stellate cells ) . RAW 264 . 7 cells were induced into M1 ( 30 ng / ml LPS for 18 hours ) or M2 ( 20 ng / ml IL - 4 for 18 hours ) macrophages . Tubulin was used as a loading control .

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Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.


Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA ( n = 223 ) using R .

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Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.


Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Cell lysates were harvested and blotted for P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( A ) .

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Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).


Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Conditioned media ( CM ) was collected mouse IL - 6 ELISA ( B - C ) .

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Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).


NIH 3T3 ( D ) , pancreatic stellate cells ( PSC ) ( E ) and human CAF cell lines CAF - PC1 and CAF - PC2 ( F ) were treated with TGFβ ( 30 ng / ml ) and / or IL - 1α ( 1 ng / ml ) for 24 hours . CM was collected for mouse or human IL - 6 ELISA .

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NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA.


CAF - PC2 cells were treated with TGFβ ( 30 ng / ml ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . CM was collected and subjected to human IL - 6 ELISA .

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CAF-PC2 cells were treated with TGFβ (30 ng/ml) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA.