Fig 2: Loss of BARD1 enhances Ewing sarcoma cell sensitivity to PARP inhibition. A, Western blot analysis for PARP1 expression in Ewing sarcoma cells. B, p-?H2AX and DAPI immunofluorescence staining of PSaRC318, A673, and CHLA10 cells treated with DMSO or 100 nmol/L talazoparib (Tal). Cells imaged at 630× and p-?H2AX foci were quantified (bottom graphs). C, IncuCyte assay comparing the confluence of PSaRC318 cells treated with DMSO versus 100 nmol/L talazoparib over 1 week. D, qRT-PCR showing BARD1 mRNA expression in A673 or CHLA10 cells treated with control (ctsi) or BARD1 (BARD1si) siRNA. E, Western blot analysis for BARD1 expression in untreated (NT) A673 or CHLA10 cells or cells treated with Ctsi or BARD1si. F, IncuCyte monitoring of cell confluence at increasing concentrations of talazoparib versus DMSO controls in A673 and CHLA10 cells treated with Ctsi versus BARD1si. A673 and CHLA10 cell data is graphed at the 60-hour time point. Normalized expression values from densitometry analyses are included under Western blots. Experiments were completed minimally in biological triplicate. NS, not significant; *, P < 0.01. Error bars, SD.

Fig 3: BARD1 binding promotes p50 mono-ubiquitination.a Immunoblot (IB) in 293T cells expressing TopBP1ER transfected with Myc-ubiquitin (Myc-Ub) and S-tag p50wt treated with tamoxifen (TAM) for the indicated time. IP performed with S-agarose and IB with anti-Myc antibody. b Cells transfected as in A and si-BARD1 or si-control treated with TAM (4 h). Nuclear fractions were isolated and IP and IB performed as in A. H3, Histone H3. c Bard1-null mouse cells transfected with Myc-Ub, S-tag p50wt and FLAG-BARD1. IP with S-agarose and IB with anti-Myc antibody. d Bard1-null cells transfected with FLAG-BARD1, S-tag p50wt, and Myc-0KUb. IP with S-agarose and IB with anti-Myc antibody. e Bard1-null cells transfected with FLAG-BARD1, Myc-Ub, S-tag p50wt, or p50S337A. IP performed with S-agarose and IB with anti-Myc antibody. f Bard1-null cells transfected with FLAG-BARD1, Myc-Ub, and S-tag p50wt or p50DE2A. IP with S-agarose and IB with anti-Myc antibody. g 293T cells transfected with Myc-Ub, S-tag p50wt, and FLAG-BARD1 wild type or the indicated BARD1 deletion mutants. IP with S-agarose and IB with anti-Myc antibody. h HCC1937 BRCA1 mutant breast cancer cells were transfected with S-tag p50wt, Myc-Ub, HA-BRCA1, and si-BARD1 as indicated. IP with S-agarose and IB with anti-Myc antibody. i In vitro ubiquitination using the indicated 6HIS-p50 protein incubated with GST-FL-BARD1, recombinant BRCA1, and Myc-Ub in the presence of UBE1 (E1) and Ubc5c (E2). IB performed with the indicated antibodies. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.

Fig 4: FL BARD1 functions in regulating apoptosis. Phospho-p53 and p53 and ß-Actin protein levels were verified by western blotting in shBARD1 and shCTR cells, in SKNSH (24 hours post-IR) and SHSY5Y (36 hours post-IR) cell lines (A). Caspase-3 activity was evaluated in SKNSH shBARD1 and shCTR IR and V cells (B) and in SHSY5Y shBARD1 and shCTR IR and V cells (C). The asterisk is indicative of p-value = 0.05. The experiments were repeated twice.

Fig 5: BARD1 RING domain deacetylation by SIRT2 promotes BRCA1-BARD1 heterodimerization(A) coIP of GFP-BARD1 before and after 4 h of AGK2 treatment shows decreased interaction with endogenous BRCA1 in HEK293T cells.(B) coIP of HA-BRCA1 after 4 h of AGK2 treatment shows decreased interaction with GFP-BARD1 in HEK293 T cells.(C and D) coIP of GFP-BARD1 3KR and 3KQ show respectively increased and decreased pull-down of endogenous BRCA1 compared with GFP-BARD1 WT in HEK293T cells.(E–H) GFP-BARD1 3KR stabilizes endogenous BRCA1 after 4 h of cycloheximide with or without AGK2 for 0 or 4 h in HEK293T cells.(I) SIRT2 knockdown decreases the interaction between endogenous BRCA1 and GFP-BARD1 WT but not 3KR in HEK293T cells.(J and K) DR-GFP assay showing that RFP-BARD1 WT and 3KR but not 3KQ restore the HR impairment of SIRT2 depletion to near control levels in U2OS cells.(L) Model showing BARD1 RING domain deacetylation by SIRT2 promoting BRCA1-BARD1 heterodimerization, thereby facilitating stability, localization, and function in HR.For the above, mean and SD from three independent replicas are shown. *p < 0.05, **p < 0.01, and ***p < 0.005.See also Figure S5.