Fig 2: Validation of BRCA1 antibodies in MEF cells. (A) MEF cell lysates were prepared and immunoprecipitation was performed using the indicated BRCA1 antibodies pre-coupled to protein A/G beads. (B) Summary of BRCA1 specific peptides detected by MS in IP samples with two anti-BRCA1 antibodies.

Fig 3: Changes in the BRCA15382insC-BARD1 EM structure under oxidative pressure.(A) Image (left) and class averages (center) of mutated BRCA15382insC-BARD1 isolated from HCC1937 cells treated with 1 mM H2O2. Scale bar, 50 nm. Projections of the 3D structure (right) agree with the class averages. Box size of averages, 25 nm. (B) Under oxidative conditions, BRCA15382insC migrates at ~270 kDa, and BARD1 migrates at ~87 kDa according to SDS-PAGE and Western blot analysis. (C) After H2O2 treatment, the RING domain of BRCA15382insC was difficult to detect compared to wild-type BRCA1 (WT). Wild-type BRCA1 (WT-R) from treated HCC70-R cells is resistant to oxidative damage. Nuclear β-actin served as a loading control. (D) The BRCA15382insC-BARD1 structure shows a clamp-like motif with extra density adjacent to the RING domain (black circle). Scale bar, 1.5 nm. Atomic models for the RING domain [magenta; PDB code, 1JM7 (18)] and a homology model of the BRCT domain (25) fit in the molecular envelope (fig. S3 and movie S3). Difference peak (yellow) indicates the additional mass present in the hotspot region of BRCA15382insC under oxidative conditions. This additional mass was not present in the untreated BRCA15382insC-BARD1 density map (gray). The red star indicates the mutation site. Scale bar, 1.5 nm. Cross sections through the RING domain are indicated (movie S3). (E) 8-OxoG formation (red) in the nuclei (blue) of HCC1937 cells treated for 40 min with 1 mM H2O2. Untreated cells (− H2O2) had inherent 8-OxoG foci. Scale bar, 50 μm. (F) Western blots indicated unstable BRCA15382insC and BARD1 compared to RAD51 in treated HCC1937 cells. Nuclear β-actin served as a loading control.

Fig 4: CDCA5 knockdown induces the DNA damage response. (A) Immunofluorescence staining revealed increased γ-H2AX foci (red) formation in 786-O cells following CDCA4 knockdown and an increased percentage of γ-H2AX-positive cells. (B) γ-H2AX foci (red) formation increased in SN12-PM6 cells following CDCA4 knockdown and the percentage of γ-H2AX-positive cells also increased. (C) Western blot analysis of proteins related to the DNA damage response following CDCA4 knockdown in both 786-O and SN12-PM6 cell lines. Scale bars, 5 µm. Data are presented as the mean ± standard deviation. **P<0.01 and ****P<0.0001 vs. negative control. CDCA5, cell division cycle-associated 5; NC, negative control; γ-H2AX, γ-H2A histone family member X; BRCA1, breast cancer type 1.

Fig 5: SKO cells do not acquire a resistant profile while exhibiting impairment in mTOR signaling and increasing DNA-damage response. A viability assay showing that SKO cells do not acquire a resistant profile after pre-treatment with 10 µM cisplatin for 72 h, both by percent survival (A) and IC50 (B). Western blot experiment showing the increase of mTOR signaling in resistant A549 WT cells, reversed by SKO after 72 h of recovery condition (C). The graph shows bands densitometry of mTOR (D), S6K1 (E), and S6 (F). Western blot showing WT and SKO response after 30 µM cisplatin for 4 h (G). The graph shows bands densitometry of p-CHK1 (H), p-CHK2 (I), Ku70 (J), and BRCA1 (K). All data represent the mean ± SD. Statistical analysis of percent viability and clonogenic survival was accessed after 2-way ANOVA followed by Bonferroni's post-test and IC50 was assessed after 2-way ANOVA followed by Tukey's post-test. Statistical of western blot was assessed after 1-way ANOVA followed by Tukey's post-test (*P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0001). Raw data are provided in Supplementary Fig. S6.