( G - H ) qPCR gene expression analysis of miRNA target genes Mapk3 , Notch1 , Dicer , Tab2 , Sox2 , Calm2 , Smad2 , Smad5 , Dnmt3 and Irak1 ( G ) and inflammatory genes Il - 1β , Tnf , Il - 6 , iNos and iκbα ( H ) in brain tissue from LPS - injected mice icv injected with vehicle ( black ) or GW4869 ( grey ; n = 7 ) . Data are displayed as mean ± SEM and analyzed by Student ' s t - test . Significance levels are indicated on the graphs : * , 0 . 01 ≤ P < 0 . 05 .

O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-H) qPCR gene expression analysis of miRNA target genes Mapk3, Notch1, Dicer, Tab2, Sox2, Calm2, Smad2, Smad5, Dnmt3 and Irak1 (G) and inflammatory genes Il-1β, Tnf, Il-6, iNos and iκbα (H) in brain tissue from LPS-injected mice icv injected with vehicle (black) or GW4869 (grey; n=7). Data are displayed as mean ± SEM and analyzed by Student's t-test. Significance levels are indicated on the graphs: *, 0.01 ≤ P < 0.05.


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 ) .

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

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 ) .

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 .

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

Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm.


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

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

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 .

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

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 ) .

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

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

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 ) .

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

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

B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY 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 ) .

B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-EXP_ASSAY 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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O

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 ) .

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

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 ) .

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

B-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY 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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

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 ) .

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

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

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 ) .

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

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

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

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

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

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

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

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

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

B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY 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 ) .

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

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 .

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

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 ) .

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

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

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.


( A - B ) qRT - PCR analysis of TGF ligands , TGF receptors , and TGF target expression in control and FRS2 knockdown HASMCs . ( NS : not significant compared to control , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) . - actin was used for sample loading normalization . Histogram of qRT - PCR results are representative of three independent experiments .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(A-B) qRT-PCR analysis of TGF ligands, TGF receptors, and TGF target expression in control and FRS2 knockdown HASMCs. (NS: not significant compared to control, *p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test). -actin was used for sample loading normalization. Histogram of qRT-PCR results are representative of three independent experiments.


( C ) Left : Immunoblot analysis of TGFRs , phosphorylated Smad2 ( p - Smad2 ) , and phosphorylated Smad3 ( p - Smad3 ) in control and FRS2 knockdown HASMCs . Blots are representative of four independent experiments . Right : Band intensities of p - Smad2 , p - Smad3 , TGFR1 , and TGFR2 were normalized to Smad2 / 3 or GAPDH and expressed as a fraction of a control value . Results are expressed as means ± SD ( * p < 0 . 05 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Left: Immunoblot analysis of TGFRs, phosphorylated Smad2 (p-Smad2), and phosphorylated Smad3 (p-Smad3) in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of p-Smad2, p-Smad3, TGFR1, and TGFR2 were normalized to Smad2/3 or GAPDH and expressed as a fraction of a control value. Results are expressed as means ± SD (*p<0.05; ***p<0.001 compared to control; unpaired two-tailed Student's t test).


( A ) Left : Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs . Blots are representative of four independent experiments . Right : Band intensities of SM - actin , SM22 , and SM - calponin were normalized to - tubulin and expressed as a fraction of a control value . Results are expressed as means ± SD ( * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test ) .

O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Left: Immunoblot analysis of smooth muscle marker gene expression in control and FRS2 knockdown HASMCs. Blots are representative of four independent experiments. Right: Band intensities of SM -actin, SM22, and SM-calponin were normalized to -tubulin and expressed as a fraction of a control value. Results are expressed as means ± SD (**p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test).


( B ) qRT - PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs . ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 compared to control ; unpaired two - tailed Student ' s t test . N = 3 ) . - actin was used for sample loading normalization .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O

(B) qRT-PCR analysis of SMC transcription factor gene expression in control and FRS2 knockdown HASMCs. (*p<0.05; **p<0.01; ***p<0.001 compared to control; unpaired two-tailed Student's t test. N=3). -actin was used for sample loading normalization.


( C ) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs ( * p < 0 . 05 compared to control ; unpaired two - tailed Student ' s t test . N = 3 ) .

O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O 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) Collagen gel contraction assays were used to determine the contractile ability of control or FRS2 knockdown HASMCs (*p<0.05 compared to control; unpaired two-tailed Student's t test. N=3).


( D - F ) Upper panels : Immunoblots of smooth muscle markers , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 ( 10 m ) , TGFR2 or Smad2 shRNA lentiviruses . Blots are representative of three independent experiments . Bottom panels : Band intensities of SM - calponin and p - Smad2 were normalized to - tubulin , HSP90 , or Smad2 and expressed as a fraction of a control value .

O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

(D-F) Upper panels: Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs treated with SB431542 (10 m), TGFR2 or Smad2 shRNA lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to -tubulin, HSP90, or Smad2 and expressed as a fraction of a control value.


( A ) Quantitative real - time PCR analysis of mature let - 7 family in control and FRS2 knockdown HASMCs . SNORD47 was used to normalize the variability in template loading . Histogram of qRT - PCR results are three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(A) Quantitative real-time PCR analysis of mature let-7 family in control and FRS2 knockdown HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.


( B ) Upper panel : Immunoblots of SM - calponin , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs transduced with or without let - 7b lentiviruses . Blots are representative of three independent experiments . Bottom panels : Band intensities of SM - calponin and p - Smad2 were normalized to GAPDH or Smad2 and expressed as a fraction of a control value .

O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(B) Upper panel: Immunoblots of SM-calponin, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs transduced with or without let-7b lentiviruses. Blots are representative of three independent experiments. Bottom panels: Band intensities of SM-calponin and p-Smad2 were normalized to GAPDH or Smad2 and expressed as a fraction of a control value.


( C ) Phase - contrast and immunofluorescence staining of smooth muscle markers ( red ) in HASMCs . Nuclei were counterstained with DAPI ( blue ) . Scale bar : 12 m . Images are representative of three independent experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O B-CELL_TYPE O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(C) Phase-contrast and immunofluorescence staining of smooth muscle markers (red) in HASMCs. Nuclei were counterstained with DAPI (blue). Scale bar: 12 m. Images are representative of three independent experiments.


( D ) HASMCs were cultured in the growth medium ( M231 + SMGS ) at day 0 then switched from growth conditions to differentiation medium ( M231 + SMDS ) for 8 days . Quantitative real - time PCR analysis of mature let - 7 family in HASMCs . SNORD47 was used to normalize the variability in template loading . Histogram of qRT - PCR results are three independent experiments .

O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(D) HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 8 days. Quantitative real-time PCR analysis of mature let-7 family in HASMCs. SNORD47 was used to normalize the variability in template loading. Histogram of qRT-PCR results are three independent experiments.


( E ) Control and FRS2 knockdown HASMCs were cultured in the growth medium ( M231 + SMGS ) at day 0 then switched from growth conditions to differentiation medium ( M231 + SMDS ) for 6 days with or without let - 7b lentiviruses . Immunoblots of smooth muscle markers , phosphorylated Smad2 ( p - Smad2 ) , and TGFR1 expression in control and FRS2 knockdown HASMCs with or without let - 7b lentiviruses . Blots are representative of three independent experiments .

O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O

(E) Control and FRS2 knockdown HASMCs were cultured in the growth medium (M231 + SMGS) at day 0 then switched from growth conditions to differentiation medium (M231 + SMDS) for 6 days with or without let-7b lentiviruses. Immunoblots of smooth muscle markers, phosphorylated Smad2 (p-Smad2), and TGFR1 expression in control and FRS2 knockdown HASMCs with or without let-7b lentiviruses. Blots are representative of three independent experiments.


( A ) Coronary arteries dissected from the human heart . Left main ( LM ) , left anterior descending ( LAD ) , and left circumflex ( LCX ) branches . Scale bar : 1 cm .

O O O B-TISSUE I-TISSUE O O O B-ORGANISM B-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O

(A) Coronary arteries dissected from the human heart. Left main (LM), left anterior descending (LAD), and left circumflex (LCX) branches. Scale bar: 1 cm.


( B ) Elastic - Van Gieson ( EVG ) staining of human coronary arteries demonstrating various degrees of atherosclerosis .

(B) Elastic-Van Gieson (EVG) staining of human coronary arteries demonstrating various degrees of atherosclerosis.


( C - D ) Representative images of immunofluorescence staining for CD31 ( green ) and SM - actin ( red ) or SM - MHC ( red ) in No / mild , moderate , and severe disease human left main coronary arteries . No : no - disease . Nuclei were stained with DAPI ( blue ) . ( b ) & amp ; ( d ) & amp ; ( f ) are high - magnification view from ( a ) & amp ; ( c ) & amp ; ( e ) . Images are representative of ten No / mild , nine moderate and ten severe disease human left main coronary artery samples . L : lumen . Scale bar : 16 m .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O

(C-D) Representative images of immunofluorescence staining for CD31 (green) and SM -actin (red) or SM-MHC (red) in No/mild, moderate, and severe disease human left main coronary arteries. No: no-disease. Nuclei were stained with DAPI (blue). (b)&(d)&(f) are high-magnification view from (a)&(c)&(e). Images are representative of ten No/mild, nine moderate and ten severe disease human left main coronary artery samples. L: lumen. Scale bar: 16 m.


( E & amp ; G ) Representative images of immunofluorescence staining for p - FGFR1 ( red ) or FGFR1 ( red ) in the same patient cohort . Nuclei were counter - stained with DAPI ( blue ) . Scale bar : 16 m .

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

(E&G) Representative images of immunofluorescence staining for p-FGFR1 (red) or FGFR1 (red) in the same patient cohort. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m.


( F & amp ; H ) Percentage of medial p - FGFR1 + SMC and FGFR1 + SMC ( * * * p < 0 . 001 compared to No / mild disease , NS : not significant compared to No / mild disease ; one - way ANOVA with Newman - Keuls post hoc test for multiple comparison correction ) .

O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F&H) Percentage of medial p-FGFR1+ SMC and FGFR1+ SMC (***p<0.001 compared to No/mild disease, NS: not significant compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction).


Figure 5 . TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis . ( A & C & E ) Representative images of immunofluorescence staining for TGF ( red ) , p - Smad2 ( red ) , or p - Smad3 ( red ) from patients with No / mild , moderate , or severe disease . Nuclei were counter - stained with DAPI ( blue ) . Scale bar : 16 m . ( B & D & F ) Medial TGF area and percentage of medial p - Smad2 , and p - Smad3 positive SMCs ( * * * p < 0 . 001 compared to No / mild disease ; one - way ANOVA with Newman - Keuls post hoc test for multiple comparison correction ) . A full table of p - values for this figure is shown in Appendix Table S1 .

O O O B-GENEPROD O O O O O O O B-ORGANISM O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5. TGFβ signaling activity in smooth muscle cells in human left main coronary arteries with various degrees of atherosclerosis. (A&C&E) Representative images of immunofluorescence staining for TGF (red), p-Smad2 (red), or p-Smad3 (red) from patients with No/mild, moderate, or severe disease. Nuclei were counter-stained with DAPI (blue). Scale bar: 16 m. (B&D&F) Medial TGF area and percentage of medial p-Smad2, and p-Smad3 positive SMCs (***p<0.001 compared to No/mild disease; one-way ANOVA with Newman-Keuls post hoc test for multiple comparison correction). A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Dissected mouse aorta demonstrating lipid - rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe - / - mice . ( b ) & ( d ) Cross - section of brachiocephalic artery from ( a ) & ( c ) stained with Oil Red O . L : lumen . Scale bar : 4 mm . 3 mice per group .

O O O O B-ORGANISM B-TISSUE O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-ORGANISM O O O

(A) Dissected mouse aorta demonstrating lipid-rich plaques in brachiocephalic artery after 4 months of high fat diet compared to the normal diet in Apoe-/- mice. (b) & (d) Cross-section of brachiocephalic artery from (a) & (c) stained with Oil Red O. L: lumen. Scale bar: 4 mm. 3 mice per group.


( B ) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti - SM - actin , anti - Notch3 , and anti - SM - MHC antibodies . Nuclei were counterstained with DAPI ( blue ) . L : lumen . Scale bar : 62 m . 3 mice per group .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O O O B-ORGANISM O O O

(B) Histological analysis of mouse normal artery or atherosclerotic plaque in brachiocephalic artery with anti-SM -actin, anti-Notch3, and anti-SM-MHC antibodies. Nuclei were counterstained with DAPI (blue). L: lumen. Scale bar: 62 m. 3 mice per group.


( C - F ) Analysis of brachiocephalic artery of Apoe - / - mice maintained for 4 months on either normal or high fat diet using anti - CD31 ( green ) , anti - p - FGFR1 ( red ) , anti - FGFR1 ( red ) , anti - p - Smad2 ( red ) , and anti - p - Smad3 ( red ) antibodies . Nuclei counterstained with DAPI ( blue ) . Scale bar : 62 m . L : lumen . M : Media . 6 mice per group .

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

(C-F) Analysis of brachiocephalic artery of Apoe-/- mice maintained for 4 months on either normal or high fat diet using anti-CD31 (green), anti-p-FGFR1 (red), anti-FGFR1 (red), anti-p-Smad2 (red), and anti-p-Smad3 (red) antibodies. Nuclei counterstained with DAPI (blue). Scale bar: 62 m. L: lumen. M: Media. 6 mice per group.


( G - J ) Quantification of the number of medial smooth muscle cells expressing p - FGFR1 , FGFR1 , p - Smad2 , and p - Smad3 ( * * * p < 0 . 001 compared to ND , NS : not significant compared to ND ; unpaired two - tailed Student ' s t test . ) . ND : Normal diet . HFD : High fat diet . A full table of p - values for this figure is shown in Appendix Table S1 .

O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(G-J) Quantification of the number of medial smooth muscle cells expressing p-FGFR1, FGFR1, p-Smad2, and p-Smad3 (***p<0.001 compared to ND, NS: not significant compared to ND; unpaired two-tailed Student's t test.). ND: Normal diet. HFD: High fat diet. A full table of p-values for this figure is shown in Appendix Table S1.


( A ) Representative photomicrographs of Oil Red O - stained atherosclerotic lesions in the aortic arch , of Apoe - / - or Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet . 3 mice per group . Scale bar : 5 mm .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O

(A) Representative photomicrographs of Oil Red O-stained atherosclerotic lesions in the aortic arch, of Apoe-/- or Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet. 3 mice per group. Scale bar: 5 mm.


( B ) ( Left ) Microphotographs of aortas ( en face ) from Apoe - / - and Frs2SMCKO / Apoe - / - mice after 16 weeks of high fat diet after staining with Oil Red O . ( Right ) Lesion area quantification . All data shown as mean ± SD . ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) . 12 mice per group .

O O O O O O B-EXP_ASSAY O B-TISSUE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM O O O

(B) (Left) Microphotographs of aortas (en face) from Apoe-/- and Frs2SMCKO/Apoe-/- mice after 16 weeks of high fat diet after staining with Oil Red O. (Right) Lesion area quantification. All data shown as mean ± SD. (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test). 12 mice per group.


( C - D ) Representative cross - sections of brachiocephalic arteries Apoe - / - and Frs2SMCKO / Apoe - / - mice stained with hematoxylin and eosin ( H & amp ; E ) ( C ) and Movat ( D ) . ( b ) & amp ; ( d ) are high - magnification view from ( a ) & amp ; ( c ) . NC : necrotic core . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

O O O O O O O O O O B-TISSUE I-TISSUE B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O

(C-D) Representative cross-sections of brachiocephalic arteries Apoe-/- and Frs2SMCKO/Apoe-/- mice stained with hematoxylin and eosin (H&E) (C) and Movat (D). (b)&(d) are high-magnification view from (a)&(c). NC: necrotic core. Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( E ) Histological analysis of atheosclerotic plaque with anti - Ki67 antibody . Nuclei were counterstained with DAPI ( blue ) . ( b ) & ( d ) are high - magnification images from ( a ) & ( c ) . Scale bar : 62 m ( low - magnification images ) ; 10 m ( high - magnification images ) . Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O

(E) Histological analysis of atheosclerotic plaque with anti-Ki67 antibody. Nuclei were counterstained with DAPI (blue). (b)&(d) are high-magnification images from (a)&(c). Scale bar: 62 m (low-magnification images); 10 m (high-magnification images). Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12.


( F ) Quantification of plaque cellularity ; Apoe - / - mice N = 9 , Frs2SMCKO / Apoe - / - mice N = 12 ( * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(F) Quantification of plaque cellularity; Apoe-/- mice N=9, Frs2SMCKO/Apoe-/- mice N=12 (***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( G ) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe - / - and Frs2SMCKO / Apoe - / - mice . ( * p < 0 . 05 , * * p < 0 . 01 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(G) Quantifications of the extent of fibrous cap and necrotic areas in brachiocephalic artery of Apoe-/- and Frs2SMCKO/Apoe-/- mice. (*p<0.05, **p<0.01 compared to Apoe-/-; unpaired two-tailed Student's t test).


( H ) Measurement of Ki67 + cells ( * p < 0 . 05 , * * * p < 0 . 001 compared to Apoe - / - ; unpaired two - tailed Student ' s t test ) .

O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O

(H) Measurement of Ki67+ cells (*p<0.05, ***p<0.001 compared to Apoe-/-; unpaired two-tailed Student's t test).


( D ) RipAC overexpression inhibits flg22 - triggered MAPK activation in Arabidopsis . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points . Immunoblots were analysed using anti - pMAPK and anti - RipAC antibodies . Coomassie brilliant blue ( CBB ) staining and a non - specific band were used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O B-GENEPROD O O B-GENEPROD O O O O O B-ORGANISM O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O

(D) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( A - B ) Interaction between RipAC and SlPUB4 / AtPUB4 was confirmed by Split - LUC assay qualitatively ( A ) and quantitatively ( B ) in Nicotiana benthamiana . Values are means ± SE ( n = 8 biological replicates ) . Asterisks indicate significant differences compared to RipAC - nLUC / cLUC - AtPIP2A negative control ( Student ' s t test , * * * * p < 0 . 0001 ) . RLU : relative luminescence units .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-ORGANISM O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O 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) Interaction between RipAC and SlPUB4/AtPUB4 was confirmed by Split-LUC assay qualitatively (A) and quantitatively (B) in Nicotiana benthamiana. Values are means ± SE (n=8 biological replicates). Asterisks indicate significant differences compared to RipAC-nLUC/cLUC-AtPIP2A negative control (Student's t test, **** p<0.0001). RLU: relative luminescence units.


( C ) Co - immunoprecipitation of SlPUB4 / AtPUB4 - GFP and RipAC - nLUC after co - expression in N . benthamiana . Two days after Agrobacterium infiltration , the plant tissues were collected and then subjected to anti - GFP immunoprecipitation . Immunoblots were analyzed using anti - LUC and anti - GFP antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O

(C) Co-immunoprecipitation of SlPUB4/AtPUB4-GFP and RipAC-nLUC after co-expression in N. benthamiana. Two days after Agrobacterium infiltration, the plant tissues were collected and then subjected to anti-GFP immunoprecipitation. Immunoblots were analyzed using anti-LUC and anti-GFP antibodies. Molecular weight (kDa) marker bands are indicated for reference.


( E ) flg22 - triggered MAPK activation in pub4 T - DNA mutants . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots . Immunoblots were analyzed using anti - pMAPK and anti - FLS2 antibodies . Coomassie brilliant blue ( CBB ) staining was used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(E) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( B ) Co - immunoprecipitation of PUB4 and BIK1 in N . benthamiana . PUB4 - GFP or GFP - LTI6b were transiently co - expressed with BIK1 - HA in the leaves of N . benthamiana . After treatment with mock or 1 μM flg22Paer for 10 min , total proteins ( input ) were extracted and subjected for immunoprecipitation with anti - GFP beads .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O

(B) Co-immunoprecipitation of PUB4 and BIK1 in N. benthamiana. PUB4-GFP or GFP-LTI6b were transiently co-expressed with BIK1-HA in the leaves of N. benthamiana. After treatment with mock or 1 μM flg22Paer for 10 min, total proteins (input) were extracted and subjected for immunoprecipitation with anti-GFP beads.


( C ) Co - immunoprecipitation of PUB4 with PRR complex members in Arabidopsis . PUB4 co - immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment , and constantly with BIK1 . Total protein extracts ( input ) from Col - 0 and PUB4 - FLAG plants were subjected to immunoprecipitation with anti - FLAG beads .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O

(C) Co-immunoprecipitation of PUB4 with PRR complex members in Arabidopsis. PUB4 co-immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment, and constantly with BIK1. Total protein extracts (input) from Col-0 and PUB4-FLAG plants were subjected to immunoprecipitation with anti-FLAG beads.


( D ) PUB4 directly interacts with BIK1 in vitro . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

(D) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.


RipAC does not affect PUB4 association with PRR complex members . PUB4 - FLAG , PUB4 - FLAG RipAC - GFP and Col - 0 plants were treated for 10 min with water ( as mock ) or 1 μM flg22Paer and elf18Ecol treatment . Total protein extracts ( input ) were subjected for immunoprecipitation with anti - FLAG beads . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference . Data information : The experiment was performed 3 times with similar results .

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

RipAC does not affect PUB4 association with PRR complex members. PUB4-FLAG, PUB4-FLAG RipAC-GFP and Col-0 plants were treated for 10 min with water (as mock) or 1 μM flg22Paer and elf18Ecol treatment. Total protein extracts (input) were subjected for immunoprecipitation with anti-FLAG beads. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Data information: The experiment was performed 3 times with similar results.


( A ) PUB4 has auto - ubiquitination activity in vitro . PUB4 autoubiquitination assay using His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) and MBP - PUB4 or MBP - PUB4 Cys239Ala inactive mutant reconstituted in bacteria . Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography ( IMAC ) . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates autoubiquitinated PUB4 .

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

(A) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.


( B ) PUB4 ubiquitinates non - active BIK1 , and does not ubiquitinate active BIK1 . Operon containing His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) , and MBP - PUB4 were co - transformed with GST , GST - BIK1 , or GST - BIK1 kinase dead ( BIK1 - KD ) in the presence of PUB4 . Ubiquitinated products were purified under denaturing conditions by IMAC , and modified proteins further enriched by GSH chromatography . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates the ubiquitination products .

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

(B) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.


( A ) Analysis of BIK1 - HA protein accumulation in BIK1 - HA + / - , pub4 - / - BIK1 - HA + / - , and PUB4 - FLAG + / - BIK1 - HA + / - in 4 - to 5 - week - old plants . Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132 , 50 mM CHX , and for 10 min with water ( as mock ) or 1 mM flg22Paer . Proteins were extracted in SDS buffer and analysed by western blot . BAK1 was used as a loading control . Numbers correspond to the quantitation of the BIK1 - HA band , normalized to the quantitation of the BAK1 control band in the same sample .

O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O

(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.


( B ) BIK1 - HA accumulation is reduced in PUB4 - FLAG + / - BIK1 - HA + / - line compared to BIK1 - HA + / - line in the absence of PAMP treatment . Total protein extracts were analysed by western blot . Tubulin was used as a loading control .

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

(B) BIK1-HA accumulation is reduced in PUB4-FLAG+/- BIK1-HA+/- line compared to BIK1-HA+/- line in the absence of PAMP treatment. Total protein extracts were analysed by western blot. Tubulin was used as a loading control.


( E ) BIK1 - HA accumulation is reduced in RipAC - GFP / BIK1 - HA line compared to Col - 0 / BIK1 - HA line . Total protein extracts were analysed by western blot . Actin was used as a loading control .

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

(E) BIK1-HA accumulation is reduced in RipAC-GFP/BIK1-HA line compared to Col-0/BIK1-HA line. Total protein extracts were analysed by western blot. Actin was used as a loading control.


D ) Cells expressing empty vector ( EV ) , or PRKAA1 - myc , PRKAB2 and PRKAG2 - FLAG ( AMPK ) alone or with myc - Ulk1 WT ( ULK ) were cultured in full medium ( F ) , EBSS ( St ) , or serum - free medium ( SFM ) alone or in the presence of 991 ( 1µM ) or MRT68921 ( 1μM ) for 90 minutes as indicated . 2 % Loading control and pull down samples were analysed by Western blot .

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

D) Cells expressing empty vector (EV), or PRKAA1-myc, PRKAB2 and PRKAG2-FLAG (AMPK) alone or with myc-Ulk1 WT (ULK) were cultured in full medium (F), EBSS (St), or serum-free medium (SFM) alone or in the presence of 991 (1µM) or MRT68921 (1μM) for 90 minutes as indicated. 2% Loading control and pull down samples were analysed by Western blot.


A ) VPS15 - ZZ was co - expressed with Ulk1 and Ulk2 wild type ( WT ) or kinase inactive ( KI ) before analysis by Western blot . An electrophoretic mobility shift in VPS15 - ZZ was noted with WT only .

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

A) VPS15-ZZ was co-expressed with Ulk1 and Ulk2 wild type (WT) or kinase inactive (KI) before analysis by Western blot. An electrophoretic mobility shift in VPS15-ZZ was noted with WT only.


A ) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot . LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA - derived clones .

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

A) Lysates from 3 control clones and 7 VPS15 CRISPR clones were analysed by Western blot. LC3 and p62 accumulation as well as depletion of VPS34 CI components was observed in all 6 sgA-derived clones.


B ) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr , fixed and imaged for WIPI2 ( green ) . Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles ( red asterisks ) . Quantification of WIPI2 spots , mean + / - SEM , n = 3 .

O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O

B) Controls and 6 VPS15 CRISPR KO clones were starved for 1 hr, fixed and imaged for WIPI2 (green). Depletion of VPS15 was associated with a block in WIPI2 puncta formation and large vacuoles (red asterisks). Quantification of WIPI2 spots, mean +/- SEM, n=3.


D ) Wild type ( WT ) , 6SA or 6SE VPS15 - HA , or empty vector ( EV ) , were expressed in VPS15 KO or HEK293A cells . Cells were starved for 1 hr and the distributions of WIPI2 ( green ) , HA ( red ) and LC3 ( blue ) were assessed , mean + / - SEM , n = 4 .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O

D) Wild type (WT), 6SA or 6SE VPS15-HA, or empty vector (EV), were expressed in VPS15 KO or HEK293A cells. Cells were starved for 1 hr and the distributions of WIPI2 (green), HA (red) and LC3 (blue) were assessed, mean +/- SEM, n=4.


D ) VPS34 CI components expressed with Ulk1 1 - 427 WT or KI as indicated . After starvation for 1 hr in the presence or absence of MRT68921 ( ULK inhibitor ) , cells were lysed and VPS34 CI coimmunoprecipitated via ATG14 - ZZ . VPS15 S861 phosphorylation was increased in the presence of active Ulk1 . VPS15 pS861 / total VPS15 was quantified , mean + / - SEM , n = 3 .

O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

D) VPS34 CI components expressed with Ulk1 1-427 WT or KI as indicated. After starvation for 1 hr in the presence or absence of MRT68921 (ULK inhibitor), cells were lysed and VPS34 CI coimmunoprecipitated via ATG14-ZZ. VPS15 S861 phosphorylation was increased in the presence of active Ulk1. VPS15 pS861/total VPS15 was quantified, mean +/- SEM, n=3.


E ) HEK293A were transfected with VPS34 CII components and Ulk1 1 - 427 WT or KI and treated as indicated before VPS34 CII co - immunopurification via UVRAG - ZZ . Samples were analysed by Western blot as in D , revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII . Quantifications show mean of 4 independent experiments + / - SEM , * * * p < 0 . 001 [ one - tailed ANOVA ] .

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

E) HEK293A were transfected with VPS34 CII components and Ulk1 1-427 WT or KI and treated as indicated before VPS34 CII co-immunopurification via UVRAG-ZZ. Samples were analysed by Western blot as in D, revealing that Ulk1 can similarly phosphorylate VPS15 at S861 when incorporated into CII. Quantifications show mean of 4 independent experiments +/-SEM, ***p<0.001 [one-tailed ANOVA].


A ) HEK293A , VPS15 KO control and stably rescued VPS15 KOs ( sgA - B3 ) were starved for 1 hr before WIPI2 ( green ) , LC3 ( red ) and DNA ( Hoechst , blue ) were visualised . Quantification of WIPI2 puncta number per cell , mean + / - SEM , n = 5 .

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

A) HEK293A, VPS15 KO control and stably rescued VPS15 KOs (sgA-B3) were starved for 1 hr before WIPI2 (green), LC3 (red) and DNA (Hoechst, blue) were visualised. Quantification of WIPI2 puncta number per cell, mean +/-SEM, n=5.


B ) Stably rescued VPS15 KO ( sgA - B3 ) , HEK293A and VPS15 KO control cells were starved for 1 hr with ( SB ) or without ( St ) 100nM Bafilomycin A1 , or cultured in full media ( F ) . Quantification shows LC3II / Actin , mean + / - SEM , n = 5 .

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

B) Stably rescued VPS15 KO (sgA-B3), HEK293A and VPS15 KO control cells were starved for 1 hr with (SB) or without (St) 100nM Bafilomycin A1, or cultured in full media (F). Quantification shows LC3II/Actin, mean +/-SEM, n=5.


A ) HEK293A were treated with VPS34 - IN1 ( 1μM ) alone or in the presence of MRT68921 ( 1μM ) for the indicated time . Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot . Identical samples were loaded in parallel for VPS34 , ATG13 , PRKAB2 analysis , indicated by dashed line .

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

A) HEK293A were treated with VPS34-IN1 (1μM) alone or in the presence of MRT68921 (1μM) for the indicated time. Untreated VPS15 KOs were included as a positive control and lysates analysed by Western blot. Identical samples were loaded in parallel for VPS34, ATG13, PRKAB2 analysis, indicated by dashed line.


E ) HEK293A and VPS15 KO cells were fed , or starved for 2 hrs before fixation , and labelling with ULK1 ( green ) , ATG9A ( red ) and GM130 ( blue ) . Dashed boxes show magnified regions of interest .

O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O

E) HEK293A and VPS15 KO cells were fed, or starved for 2 hrs before fixation, and labelling with ULK1 (green), ATG9A (red) and GM130 (blue). Dashed boxes show magnified regions of interest.


B ) HEK293A were transfected with siRNAs targeting ATG14 , UVRAG or p62 , or with non - targeting siRNA ( RISC - free , RF ) and cells were treated with VPS34 - IN1 ( IN1 ; 1μM ) for 0 , 4 or 18 hrs as indicated . Quantification of ATG13 pS318 / Total ATG13 , mean + / - SEM , n = 3 . Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis , indicated by dashed line .

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

B) HEK293A were transfected with siRNAs targeting ATG14, UVRAG or p62, or with non-targeting siRNA (RISC-free, RF) and cells were treated with VPS34-IN1 (IN1; 1μM) for 0, 4 or 18 hrs as indicated. Quantification of ATG13 pS318/Total ATG13, mean +/- SEM, n=3. Identical samples were loaded onto 2 separate SDS PAGE gels for Western blot analysis, indicated by dashed line.


C ) Cells transfected as in B were treated with IN1 for 18 hours as indicated , fixed and labelled for ubiquitin ( green ) , FIP200 ( red ) and p62 ( blue ) . FIP200 - positive bodies were quantified from 4 independent experiments with mean + / - SEM plotted . Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested .

O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O

C) Cells transfected as in B were treated with IN1 for 18 hours as indicated, fixed and labelled for ubiquitin (green), FIP200 (red) and p62 (blue). FIP200-positive bodies were quantified from 4 independent experiments with mean +/- SEM plotted. Cells transfected with ATG14 siRNA generated significantly more FIP200 bodies per cell than all other conditions tested.


Validation of upregulated lncRNAs ( ΔCT ) in PBMC - derived M2 - like macrophages ( stimulated with IL - 4 stimulated for 24 hours , M2 ) . n = 6 biological replicates , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with M1 - like macrophages ( LPS / IFNɣ - stimulated for 24 hours , M1 ) .

O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-CELL_TYPE O O

Validation of upregulated lncRNAs (ΔCT) in PBMC-derived M2-like macrophages (stimulated with IL-4 stimulated for 24 hours, M2). n = 6 biological replicates, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with M1-like macrophages (LPS/IFNɣ-stimulated for 24 hours, M1).


Representative images of fluorescence in - situ hybridization of ADPGK - AS1 ( magenta ) and nuclei ( blue ) in IL - 4 stimulated M2 - like macrophages . β - actin served as the cytoplasmic control and MALAT1 as the nuclear control . Scale bar = 10 µm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O

Representative images of fluorescence in-situ hybridization of ADPGK-AS1 (magenta) and nuclei (blue) in IL-4 stimulated M2-like macrophages. β-actin served as the cytoplasmic control and MALAT1 as the nuclear control. Scale bar = 10 µm.


RNA expression analysis of ADPGK - AS1 , 47S ribosomal RNA ( nuclear control ) , and β - Tubulin ( cytoplasmic control ) in IL - 4 stimulated M2 - like macrophages after subcellular fractionation . n = 3 independent experiments . Data represent the percentage of total detected RNA .

O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

RNA expression analysis of ADPGK-AS1, 47S ribosomal RNA (nuclear control), and β-Tubulin (cytoplasmic control) in IL-4 stimulated M2-like macrophages after subcellular fractionation. n = 3 independent experiments. Data represent the percentage of total detected RNA.


Subcellular fractionation of IL - 4 - stimulated M2 - like macrophages and isolation and analysis of ADPGK - AS1 abundance in the cytoplasmic and mitochondrial fractions The mitochondrial encoded RNA mtATP6 served as the mitochondrial control and β - tubulin as the cytoplasmic control . n = 3 independent experiments , Data represent the percentage of total detected RNA .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

Subcellular fractionation of IL-4-stimulated M2-like macrophages and isolation and analysis of ADPGK-AS1 abundance in the cytoplasmic and mitochondrial fractions The mitochondrial encoded RNA mtATP6 served as the mitochondrial control and β-tubulin as the cytoplasmic control. n = 3 independent experiments, Data represent the percentage of total detected RNA.


( A ) Validation of the ADPGK - AS1 - interacting protein MRPL35 by RNA immunoprecipitation ( RIP ) with followed by qPCR for ADPGK - AS1 . n = 4 independent experiments , * * * p ≤ 0 . 001 compared with IgG control .

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

(A) Validation of the ADPGK-AS1-interacting protein MRPL35 by RNA immunoprecipitation (RIP) with followed by qPCR for ADPGK-AS1. n = 4 independent experiments, ***p ≤ 0.001 compared with IgG control.


( B , C ) Co - staining for ADPGK - AS1 ( magenta ) and MRPL35 or MRPL15 ( yellow ) with respective fluorescence intensity across co - stained macrophages ( see white line ) for ADPGK - AS1 and MRPL35 or MRPL15 . Scale bar = 10 µm ( left ) , 5 µm ( right ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(B, C) Co-staining for ADPGK-AS1 (magenta) and MRPL35 or MRPL15 (yellow) with respective fluorescence intensity across co-stained macrophages (see white line) for ADPGK-AS1 and MRPL35 or MRPL15. Scale bar = 10 µm (left), 5 µm (right).


( E ) Number of M1 - like or M2 - like macrophages based on TMA data for lung cancer patients with either high ( above median ) or low ( below median ) expression of MRPL35 .

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(E) Number of M1-like or M2-like macrophages based on TMA data for lung cancer patients with either high (above median) or low (below median) expression of MRPL35.


( F , G ) Kaplan - Meier curve estimate of disease - free survival ( DFS ) or overall survival ( OS ) of patients with high ( above median ) or low ( below median ) expression of MRPL35 in M2 - like macrophages . n = 200 biological replicates .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O

(F, G) Kaplan-Meier curve estimate of disease-free survival (DFS) or overall survival (OS) of patients with high (above median) or low (below median) expression of MRPL35 in M2-like macrophages. n = 200 biological replicates.


( C , Representative images and quantification of mitochondria by MitoTracker ( red ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . Scale bar = 200 µm ( left ) , 100 µm ( right , magnification ) .

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

(C, Representative images and quantification of mitochondria by MitoTracker (red) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).


D ) Representative images and quantification of mitochondria by TOM20 ( green ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . Scale bar = 200 µm ( left ) , 100 µm ( right , magnification ) .

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

D) Representative images and quantification of mitochondria by TOM20 (green) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm (left), 100 µm (right, magnification).


( E ) Representative immunoblot for mitochondrial protein expression ( TOM20 , αb - crystallin , PCG - 1 , MRPL35 , cytochrome C ) in THP1 control and ADPGK - AS1 OE cells . n = 4 independent experiments .

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

(E) Representative immunoblot for mitochondrial protein expression (TOM20, αb-crystallin, PCG-1, MRPL35, cytochrome C) in THP1 control and ADPGK-AS1 OE cells. n = 4 independent experiments.


( G ) Gene expression analysis ( fold change ) of mitochondrially encoded genes ( ND1 , ND2 , ND4 , ATP6 , ATP8 , CO1 , CO2 , CyB ) after isolation of mtDNA from THP1 control and ADPGK - AS1 OE cells . n = 3 independent experiments . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with control .

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

(G) Gene expression analysis (fold change) of mitochondrially encoded genes (ND1, ND2, ND4, ATP6, ATP8, CO1, CO2, CyB) after isolation of mtDNA from THP1 control and ADPGK-AS1 OE cells. n = 3 independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 compared with control.


I ) full oxygen consumption plot ( n = 8 ) by the seahorse assay in THP1 control and ADPGK - AS1 OE cells . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 compared with control .

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I) full oxygen consumption plot (n = 8) by the seahorse assay in THP1 control and ADPGK-AS1 OE cells. **p ≤ 0.01, ***p ≤ 0.001 compared with control.


( K ) Representative confocal fluorescence images and quantification of mitochondrial shape with the mitochondrial marker TOM20 ( yellow ) in THP1 control and ADPGK - AS1 OE cells . Nuclei were stained with DAPI ( blue ) . n = 35 cells , Scale bar = 10µm ( left ) , 5µm ( magnification , right ) .

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

(K) Representative confocal fluorescence images and quantification of mitochondrial shape with the mitochondrial marker TOM20 (yellow) in THP1 control and ADPGK-AS1 OE cells. Nuclei were stained with DAPI (blue). n = 35 cells, Scale bar=10µm (left), 5µm (magnification, right).


( L ) Transmission electron microscopy images of mitochondrial shapes in THP1 control and ADPGK - AS1 OE cells . n = 4 cells , Scale bar = 5 µm ( left ) , 1 µm ( right , magnification ) .

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

(L) Transmission electron microscopy images of mitochondrial shapes in THP1 control and ADPGK-AS1 OE cells. n = 4 cells, Scale bar = 5 µm (left), 1 µm (right, magnification).


( B ) Expression analysis of ADPGK - AS1 and MRPL35 ( fold change ) in THP1 control and ADPGK - AS1 OE macrophages transfected with a siRNA against ADPGK - AS1 , MRPL35 or negative control . n = 4 independent experiments . * * * p ≤ 0 . 001 compared with ADPGK - AS1 OE siRNA control .

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

(B) Expression analysis of ADPGK-AS1 and MRPL35 (fold change) in THP1 control and ADPGK-AS1 OE macrophages transfected with a siRNA against ADPGK-AS1, MRPL35 or negative control. n = 4 independent experiments. ***p ≤ 0.001 compared with ADPGK-AS1 OE siRNA control.


( C ) Representative images of JC - 1 monomers ( green ) and aggregates ( red ) in THP1 control and ADPGK - AS1 overexpressing macrophages transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . Scale bar = 100 µm .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O

(C) Representative images of JC-1 monomers (green) and aggregates (red) in THP1 control and ADPGK-AS1 overexpressing macrophages transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. Scale bar = 100 µm.


( E ) Representative images ( left panel ) and quantification ( right panel ) of ROS accumulation in THP1 control and ADPGK - AS1 OE cells transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . n = 7 . * * * p ≤ 0 . 001 compared to control .

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

(E) Representative images (left panel) and quantification (right panel) of ROS accumulation in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 7. ***p ≤ 0.001 compared to control.


( F ) Spare capacity ( oxygen consumption rate ) measured with the seahorse assay in THP1 control and ADPGK - AS1 OE cells transfected with a siRNA against ADPGK - AS1 , MRPL35 , or negative control . n = 3 independent experiments . * * * p ≤ 0 . 001 compared with control , § p ≤ 0 . 05 , § § § p ≤ 0 . 001 compared with ADPGK - AS1 OE siRNA negative control .

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

(F) Spare capacity (oxygen consumption rate) measured with the seahorse assay in THP1 control and ADPGK-AS1 OE cells transfected with a siRNA against ADPGK-AS1, MRPL35, or negative control. n = 3 independent experiments. ***p ≤ 0.001 compared with control, §p ≤ 0.05, §§§p ≤ 0.001 compared with ADPGK-AS1 OE siRNA negative control.


( B ) mRNA expression analysis ( ΔCT ) of macrophage markers ( TNFα , IL - 8 , CD206 , CSF - 1R ) in co - cultured PBMC - derived macrophages with tumor cells or macrophage control ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , * * p ≤ 0 . 01 compared with M0 .

O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-CELL_TYPE O O 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) mRNA expression analysis (ΔCT) of macrophage markers (TNFα, IL-8, CD206, CSF-1R) in co-cultured PBMC-derived macrophages with tumor cells or macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.


( C ) Expression of ADPGK - AS1 lncRNA in macrophages co - cultured with tumor cells ( A549 ) or macrophage controls ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , compared with M0 .

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

(C) Expression of ADPGK-AS1 lncRNA in macrophages co-cultured with tumor cells (A549) or macrophage controls (M0). n = 4 biological replicates. *p ≤ 0.05, compared with M0.


( D ) Mass spectrometry analysis of TCA cycle metabolites ( fumarate , malate , citrate , succinate ) in macrophages co - cultured with tumor cells ( A549 ) and macrophage control ( M0 ) . n = 4 biological replicates . * p ≤ 0 . 05 , * * p ≤ 0 . 01 compared with M0 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O B-CELL_LINE O O B-CELL_TYPE O O B-CELL_TYPE 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-CELL_TYPE O

(D) Mass spectrometry analysis of TCA cycle metabolites (fumarate, malate, citrate, succinate) in macrophages co-cultured with tumor cells (A549) and macrophage control (M0). n = 4 biological replicates. *p ≤ 0.05, **p ≤ 0.01 compared with M0.


( G ) Expression of IDH mRNA in A549 cells transfected with siRNA specific for IDH ( IDH KD ) or negative control . n = 5 independent experiments . * * * p ≤ 0 . 001 compared with A549 control .

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

(G) Expression of IDH mRNA in A549 cells transfected with siRNA specific for IDH (IDH KD) or negative control. n = 5 independent experiments. ***p ≤ 0.001 compared with A549 control.