Fig 2: BRCA1 protects GBM cells from replication stress-induced DSBs formation.(a) Microscopy analysis and quantification of pRPA mean signal intensity (in S phase) in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (b) Microscopy analysis and quantification of Rad51 mean signal intensity (S phase) in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (c) Microscopy analysis and quantification of 53BP1 foci count (G1 phase) in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (d) Immunoblot analysis of DDR activation in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). * Chk1Ser317; # Chk1Ser345. (e) FACS quantification of double-positive p-RPA/?H2AX GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (f) FACS quantification of double-positive PCNA/?H2AX GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (g) Microscopy analysis and quantification of ?H2AXfoci count in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (h) Comet assay and tail moment quantification of DSBs in GBM cells transduced with shCTRL or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in a–c,e–h and all data are shown as means ±s.d. and performed as technical triplicates. (*P<0.05, **P<0.005, ***P<0.005, ****P<0.0001; NS represents non-significance).

Fig 3: BRCA1 regulates RRM2 expression via E2F1 and RRM2 overexpression rescues BRCA1-loss phenotype in GBM cells.(a) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 transduced with shCTRL or shBRCA1-2/shBRCA1-4. (b) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of Sp1; AP-1 and E2F1 in comparison to BRCA1. (c) Luciferase assay of transcriptional activation of the RRM2 promoter in GBM01 after siRNA-mediated knockdown of BRCA1 and E2F1 alone or on combination. (d) Chip immunoprecipitation of E2F1 binding RRM2 promoter in GBM01 cells using primer set P1 and P2. (e) Chip immunoprecipitation of BRCA1 binding RRM2 promoter in GBM01 cells transfected with siCTRL and siE2F1 using primer set P1 and P2. (f) DNA fibre assay measuring the replication fork progression speed in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). See also Table 3. (g) Cell viability of GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). (h) Immunoblot analysis of BRCA1, p-RPA, RPA and RRM2 in GBM cells lacking BRCA1 (shBRCA1-2 and shBRCA1-4) or not (shCTRL) transfected with either control vector (pcDNA) or vector expressing RRM2 (pcDNA-RRM2). (i) Viability-based assessment of EC50 triapine concentrations in GBM01, GBM02 and GBM03 cells. (j) DNA fibre assay measuring the replication fork progression speed in GBM cells treated with DMSO or EC50 triapine. See also Table 4. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test in a–d,f,h or Student’s t test (e,i) and all data are shown as means ±s.d. and performed as technical triplicates. (*P<0.05, **P<0.005, ***P<0.005, ****P<0.0001; NS represents non-significance).

Fig 4: BRCA1 is essential for GBM cell maintenance.(a) Viability curves for GBM01, GBM02 and GBM03 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4) (7 days). (b) Quantification of apoptotic cells (% of Annexin V-positive) for GBM01, GBM02 and GBM03 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (c) Immunoblot analysis of BRCA1, caspase 3 and cleaved caspase-3 in GBM01 and GBM02 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). Tubulin was used as a loading control. (d) FACS analysis of cell cycle profile and the proliferative index (% of Edu-positive cells) in GBM01 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (e) FACS analysis of cell cycle profile and the mitotic index (% of H3Ser10-positive cells) in DMSO or nocodazole treated GBM01 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4). (f) Kaplan-Meier survival curve for NMRI nude mice intracranially injected with GBM01 or GBM03 cells transduced with shCTRL virus or two independent BRCA1-targeting shRNAs (shBRCA1-2 and shBRCA1-4 or shBRCA1-5). Statistical significance was calculated by one-way ANOVA, Tukey’s multiple comparisons test (in vitro study) and Log-rank/Mantel-Cox test (in vivo study). All data are shown as means±s.d. and performed as technical triplicates. (*P<0.05, **P<0.005, ***P<0.005, ****P<0.0001; NS represents non-significance).

Fig 5: Expression and prognostic value of BRCA1 and RRM2 in malignant gliomas.(a) IHC staining of BRCA1 in normal brain (NB), WHO gr. II, III, IV glioma. Shown are representative sections. Scale bar 50 µm. (b) Quantification of (a) BRCA1 staining. Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. (c) A survival curve for BRCA1 negative (n=32); BRCA1 low (< 14.5% of BRCA1+ cells, n=35) or BRCA1 high (>14.5% of BRCA1+ cells, n=78; median survival of 230 days) glioma patients; Log-rank P value=0.00. Median survival for BRCA1 negative and low patient is not available as >50% of patients were alive at the end of study. (d) A survival curve for BRCA1 high (>14.5% of BRCA1+ cells, n=60, median survival of 159 days) and BRCA1 low (<14.5% of BRCA1+ cells, n=15; median survival of 251 days) GBM patients. Log-rank P-value=0.293. (e) Quantification of RRM2 staining (% of positive cells). Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. (f) A survival curve for RRM2 negative (n=64) and RRM2 positive (n=81; median survival of 222 days) glioma patients, where the median RRM2-positivity is 1% and Log-rank P value=0.00. Median survival for RRM2 negative patients is not available as more than 50% of patients were alive at the end of study. (g) A survival curve for RRM2 positive (n=56, median survival of 148 days) and RRM2 negative (n=19; median survival of 320 days) GBM patients. Log-rank P value=0.23. (h) Spearman correlation test confirmed a positive correlation between the percentage of BRCA1 and RRM2-positive cells in human gliomas (our study dataset). (i) Spearman correlation test confirmed a positive correlation between BRCA1 and RRM2 mRNA expression in human gliomas (REMBRANDT dataset). (*P<0.05, **P<0.005, ***P<0.005, ****P<0.0001; NS represents non-significance).