Fig 2: USP37 regulates DDR through BLM. (A) Cells from Supplementary Figure S4A were treated with cisplatin (3 μmol/l) at the indicated time points before fixing and processed for γ-H2AX immunofluorescence. Representative γ-H2AX foci (cisplatin treatment) are shown (A) (magnification: 40×). (B, C) Quantification of γ-H2AX focus formation in (A). The graph represents mean ± 95% CI, two-tailed, unpaired t-test was applied (n = 60 in each group). (D) USP37-WT, but not S114A mutant, rescued USP37 knockdown-mediated high levels of γ-H2AX post-cisplatin treatment at the indicated time points. IB (Immunoblot) analysis of WCLs (whole-cell lysates) derived from USP37 knockdown U-2OS cells rescued with Ctrl, USP37-WT and USP37-S114A as well as empty vector (EV), which were treated with cisplatin (3 μmol/l) at indicated time points before harvesting. (E) Cells from (D) treated with cisplatin (3 μmol/l) at the indicated time points before fixing and processed for γ-H2AX immunofluorescence. Representative γ-H2AX foci (cisplatin treatment) are shown (E) (magnification: 40×). (F) Quantification of γ-H2AX focus formation in (E). The graph represents mean ± 95% CI, two-tailed, unpaired t-test was applied (n = 60 in each group). (G–I) The response of control cells, USP37 knockdown cells and USP37 knockdown U-2OS cells stably expressing the indicated constructs to cisplatin (H) or ionizing radiation (IR) (I) was assessed using colony formation assays. USP37 expression is shown in (G). Data are the mean (±SEM) of three independent experiments. Statistical significance was assessed by two-way ANOVA with Tukey's multiple comparisons test.

Fig 3: USP37 expression positively correlates with BLM levels in breast cancer tissues. (A, B) USP37 expression in normal tissue and breast carcinoma (Oncomine data). (C) GEO database showing the survival of breast cancer patients with low (n = 112) and high (n = 127) expression of USP37. Statistical analysis with the two-sided log-rank (Mantel-Cox) test revealed statistically significant differences as shown on the graph. (D) Representative images of immunohistochemical staining of USP37 and BLM in normal and breast carcinoma. Scale bars, 200 μm. (E) Quantification of USP37 and BLM protein levels in normal and breast carcinoma. (F) Correlation study of USP37 and BLM in breast carcinoma. Statistical analyses were performed with the χ2 test. R: the Pearson correlation coefficient.

Fig 4: USP37 regulates chemotherapy and radiotherapy resistance via BLM. (A) Immunoblot of indicated proteins in human breast epithelial cell line and breast carcinoma cell lines. (B) MCF7 cells stably expressing indicated constructs were lysed. Cells were lysed and cell lysates were blotted with the indicated antibodies. (C, D) Cells from (B) were treated with (C) cisplatin or (D) IR. Cell survival was performed by CCK8 assays. (E) T-47D cells stably expressing indicated constructs were lysed. Cells were lysed and cell lysates were blotted with the indicated antibodies. (F–G) Cells from (E) were treated with (F) cisplatin or (G) IR. Cell survival was performed by CCK8 assays. (H) MCF7 cells stably expressing indicated constructs were lysed. Cells were lysed and cell lysates were blotted with the indicated antibodies. (I, J) Cells from (H) were treated with (I) cisplatin or (J) IR. Cell survival was performed by CCK8 assays. The data presented are mean ± SD (n = 6). (K–M) Tumor xenograft assays were performed by subcutaneous injection of USP37 knockdown MDA-MB-231 cells stably expressing USP37 WT, S114A, and EV. Tumor growth rate in nude mice treated every other day with cisplatin (20 mg/kg) is shown (K). Tumors were dissected and recorded after euthanizing the mice (L). Mice were sacrificed after 28 days. Tumor weights were measured as shown in (M). Representative data (mean ± SEM) are shown from six mice each group by two-sided unpaired t-test.

Fig 5: USP37-promoted BLM stabilization is potentiated by DNA damage. (A) HEK293T cells exposed to cisplatin (3 μmol/l) were collected at the indicated time points and analyzed by western blotting with phospho-CHK2 as a positive control. (B) Total RNA was collected from HEK293T cells exposed to cisplatin followed by qRT-PCR analysis of BLM expression. Data represent the mean ± SD of biological triplicate experiments. (C) HEK293T cells stably expressing control (Ctrl) or USP37 shRNAs were exposed to cisplatin (3 μmol/L) and collected at the indicated time points. Expression of the indicated proteins was assessed by western blotting. (D) HEK293T cells stably expressing vector or wild type HA-USP37 were exposed to cisplatin (3 μmol/l) for 16 h and then treated with MG132 for 4 h to normalize the BLM protein level. IP with HA-tagged beads was performed. Blots were probed with the indicated antibodies. (E) MCF7 cells were exposed to cisplatin (3 μmol/l) for 16 h and then treated with MG132 for 4 h to normalize the BLM protein level. Cellular extracts were immunoprecipitated and then immunoblotted with antibodies against the indicated proteins. (F) HEK293T cells stably expressing control (Ctrl) or USP37 shRNAs were transfected with Myc-BLM and His-Ub for 36 h, cells were then untreated or treated with cisplatin (3 μmol/l) for 16 h, and then treated with MG132 for 4 h before harvest. Cell lysates were immunoprecipitated with Ni-NTA beads. Blots were probed with the indicated antibodies.