Fig 2: UBE2O-mediated cancer stemness properties in BC cells.a The indicated cells were cultured in cancer stemness medium for 2 weeks. Then, the cells were observed under an optical microscope, and the numbers of stem-like spheres formed in each group were determined in triplicate plates (upper: magnification × 100, Scale bar, 100 µm; lower: magnification × 400, Scale bar, 20 µm). b Expression levels of cancer stemness markers (CD44, ABCG2, OCT4 and MYC) were determined in MDA-MB-231sh-NC/MDA-MB-231sh-UBE2O#1 cells and MCF-7NC/MCF-7OE-UBE2O cells by western blotting. c, d The CD44 and MYC expression status of BC patients were detected by IHC (n = 100, upper: magnification × 100, lower: magnification × 400), and their correlation with UBE2O was calculated by Pearson’s correlation coefficient. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent at least three independent experiments.

Fig 3: Effects of CP on XOD activity and ABCG2 expression. (A) In vitro XOD inhibition. (B) Serum XOD activity. (C) Liver XOD activity. (D) Relative protein expression levels of hepatic XOD. (E) Relative protein expression level of renal ABCG2. (F) Relative mRNA level of hepatic XOD. (G) Relative mRNA level of renal ABCG2. The protein expression level was determined by Western blotting and the relative gene level was determined by RT-qPCR. The results are expressed as mean ± SD (n = 10). ## p < 0.01 compared with NC group; * p < 0.05, ** p < 0.01 compared with HUA group.

Fig 4: UBE2O induced proliferation, EMT and CSPs in a manner dependent on the regulation of the UBE2O/AMPKa2/mTORC1 axis in BC.a Western blotting revealed that p-Raptor expression was increased in MDA-MB-231sh-UBE2O#1cells, which indicated mTORC1 pathway inhibition. As a downstream effector of mTORC1, p-S6K expression was also decreased. The opposite results occurred in MCF-7OE-UBE2O cells. b–f AMPKa2 was knocked down in MDA-MB-231sh-NC/MDA-MB-231sh-UBE2O#1cells. Then, b scratch assays, c invasion assays, d colony formation assays, e CCK-8 assays and f sphere formation assays were performed (upper: magnification × 100, Scale bar, 100 µm; lower: magnification × 400, Scale bar, 20 µm). g Western blot assays were performed to detect the expression of EMT markers (CDH1, CDH2, vimentin and slug), CS markers (CD44, ABCG2, OCT3/4 and MYC) and mTORC1 pathway-related markers (p-Raptor and p-S6K) after AMPKa2 suppression in MDA-MB-231sh-NC/MDA-MB-231sh-UBE2O#1cells. The data are shown as the mean ± s.d. Student’s t test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001. The data represent at least three independent experiments.

Fig 5: UBE2O induced proliferation, EMT and CSPs in a manner dependent on the regulation of the UBE2O/AMPKa2/mTORC1 axis in BC.a MCF-7NC/MCF-7OE-UBE2O cells were treated with rapamycin (100 nm) for 24 h. Then, the cells were lysed, and the expression of EMT markers (CDH1, CDH2, vimentin and slug), CS markers (CD44, ABCG2, OCT3/4 and MYC) and mTORC1 pathway correlative markers was detected by western blot assays. b–f MCF-7NC/MCF-7OE-UBE2O cells were treated with rapamycin (100 nm), and b cell migration, c invasion, d, e proliferation and f CSPs (upper: magnification × 100, Scale bar, 100 µm; lower: magnification × 400, Scale bar, 20 µm) were detected. The data are shown as the mean ± s.d. Student’s t test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001. The data represent at least three independent experiments.