Fig 2: USP1 binds and stabilizes KDM4A. A, Proteins that interacted with KDM4A were identified by Co-IP and mass spectrometry assays. B, RV1 cells were transfected with Flag-KDM4A plasmids for 48 h, and lysates were subjected to immunoprecipitation with anti-KDM4A Ab. Bound proteins were analyzed by western blotting with anti-KDM4A or USP1 Abs. C, Representative fluorescence images of KDM4A and USP1 proteins in RV1 cells. Cells were seeded on coverslips for 24 h and then stained for KDM4A (red) and USP1 (green). Nuclei were visualized by DAPI staining (blue). D, E, RV1 cells were lysed and immunoprecipitated (IP) with indicated Abs. Immunocomplexes were subjected to western blotting. F, RV1 cells stably expressing control or WDR48 shRNAs were lysed and IPed with indicated Abs. Immunocomplexes were subjected to western blotting. G, RV1 cells stably expressing control or USP1 shRNAs were subjected to western blotting to examine the indicated proteins. H, Cells were untreated or treated with 10 µmol/L MG-132 and western blotting was carried out to examine the indicated protein levels. I, 293T cells were transfected with indicated constructs. Indicated proteins were analyzed by western blotting. J, RV1 cells were stably expressed control or Flag-USP1 constructs and then cycloheximide (CHX) pulse-chase assay was undertaken in cells. Right panel, protein levels of USP1 relative to ß-actin

Fig 3: Targeting USP1 promotes prostate cancer cells’ response to therapeutic agent enzalutamide. A, RV1 (left panel) or LNCAP (right panel) cells were treated with the indicated concentrations of ML323 for 48 hours and analyzed by western blotting for the indicated proteins. B, RV1 or LNCAP cells were treated with 5µmol/L ML323 for 48 h and mRNA levels of the indicated genes were detected (mean ± SEM of 3 independent experiments). *P < .05; **P < .01. C, RV1 cells were seeded at low density on the plate, and maintained for 2 wk. Numbers of colonies were counted in 5 high-power fields (mean ± SEM of 3 independent experiments). **P < .01. D, RV1 cells were stably expressed with or without Flag-KDM4A constructs and treated with either vehicle or USP1 inhibitor ML323 for 24 h. Cells were subjected to western blot to examine the indicated protein levels. E, Cells as described in (D) were treated with the indicated concentrations of enzalutamide and cell survival was determined (mean ± SD [n = 3]). F, RV1 cells stably expressing control (Ctrl) or USP1 shRNA with or without Flag-KDM4A were subjected to western blotting to detect the indicated protein levels. G, Cells as described in (F) were treated with the indicated concentrations of enzalutamide and cell survival was determined (mean ± SD [n = 3]). H, Schematic representation of how USP1 regulates androgen receptor (AR) and c-Myc activities through KDM4A

Fig 4: USP1 overexpression increases cell proliferation in a partially TAZ-dependent manner. (A) Immunoblot analysis for USP1 and TAZ in USP1 transduced MDA-MB-231, 293T and MCF7 cells. GAPDH was used as a loading control. (B) Cell proliferation assay of USP1 overexpressing MDA-MB-231 cells. (C) Quantification of clonogenicity assay after 11 days in USP1 overexpressing MDA-MB-231 cells. (D) Representative images and quantification of spheroid size in control or USP1 overexpressing MDA-MB-231 cells (Scale bar = 50 µm). (E) Immunoblot analysis of TAZ knockdown in USP1 overexpressing MDA-MB-231 cells. GAPDH was used as a loading control. (F) Cell proliferation assay for shCtrl or shTAZ in USP1 overexpressing MDA-MB-231. (G) Quantification of clonogenicity assay after 11 days for shCtrl or shTAZ in USP1 overexpressing MDA-MB-231. Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. Immunoblots were quantified and normalized to GAPDH.

Fig 5: USP1 acts as a post-translational modifier of TAZ. (A) Immunoblot analysis for USP1 and TAZ expression in USP1 knockdown MCF10A and MDA-MB-231 cells. GAPDH was used as a loading control. (B) Relative mRNA expression of USP1 and TAZ in USP1 knockdown MCF10A and MDA-MB-231 cells. (C) Immunoblot analysis for TAZ expression in MCF10A and MDA-MB-231 upon treatment with 15µM ML323. GAPDH was used as a loading control. (D) Immunoblot analysis for TAZ expression in USP1 knockdown MCF10A and MDA-MB-231 cells with MG132treatment. GAPDH was used as a loading control. (n = 2) (E) Immunoblot analysis for USP1 and TAZ in USP1 knockdown MDA-MD-231 cells with CHX treatment at various time points. GAPDH was used as a loading control. (n = 3) (F) Immunoblot analysis for USP1 and TAZ expression in USP1 overexpressing MCF7 cells with CHX treatment at various time points. GAPDH was used as a loading control. (n = 2) Data are shown as the mean ± SD. Unpaired two-tailed student t-test: * p < 0.05, ** p < 0.01, *** p < 0.001. NS: no significance. Immunoblots were quantified and normalized to GAPDH.