Fig 2: OTUD5 is overexpressed and acts as an oncogene in bladder cancer.A Representative images of OTUD5 immunohistochemistry (IHC) staining in bladder cancer tissue and adjacent normal bladder tissue samples. Scale bars, upper 500 μm, lower 100 μm. B Statistical analysis of OTUD5 expression in adjacent normal bladder tissues and bladder cancer tissues. ***P < 0.001. C, D Western blot analysis and quantitative real-time RT-PCR of OTUD5 expression in the normal human bladder cell line and bladder cancer cell lines. E Western blot analysis of OTUD5 protein expression in adjacent normal bladder tissues and cancer tissues. β-actin was used as the loading control. F. The quantitative analysis of the relative expression of OTUD5 protein. G, H Quantitative real-time RT-PCR and Western blotting analysis of OTUD5 expression in 253 J or UM-UC-14 cell lines transfected with OTUD5 shRNAs and shNC, and T24 cell line infected with OTUD5 lentivirus and negative control. 18 S was applied as the endogenous control for quantitative real-time RT-PCR, and β-actin was used as a loading control for Western blotting assay. I Colony formation assays and quantification results of 253 J and UM-UC-14 cells with OTUD5 knockdown. ***P < 0.001. J Colony formation assays and quantification results of T24 cells with OTUD5 overexpression. Vec vector. ***P < 0.001. K, L, M MTT assay was performed to detect viability in 253 J and UM-UC-14 cells with OTUD5 knockdown and T24 cells with OTUD5 overexpression. **P < 0.01. All data are presented as mean ± SD of 3 independent experiments.

Fig 3: OTUD5 knockdown combined with everolimus inhibits bladder cancer growth in vitro and in vivo.A Colony formation assays and quantification results of UM-UC-14/WT and UM-UC-14/shOTUD5 cells treated with or without everolimus (10 nM). **P < 0.01, ***P < 0.001. B The growth curves of UM-UC-14/WT and UM-UC-14/shOTUD5 cells treated with or without everolimus (10 nM). ***P < 0.001. C The growth curves of xenografts in different treatment groups. Tumor volumes were measured every three days from day 12 to day 30. ***P < 0.001. D Illustration of tumors excised from male nude mice in each treatment group. E After 30 days, the nude mice were sacrificed, and UM-UC-14 cell xenografts were weighed. **P < 0.01, ***P < 0.001. F The expressions of OTUD5, p-mTOR, RNF186 and sestrin2 in the xenograft tumors were measured by immunohistochemistry. (Scale bar, 20 µm). G The expressions of OTUD5, p-mTOR, RNF186 and sestrin2 in the xenograft tumors were measured by Western blot. β-actin was used as the loading control. n = 6 mice per treatment group, data are presented as mean ± SD.

Fig 4: OTUD5 positively regulates the mTOR signaling pathway.A Flag-OTUD5 was immunoprecipitated (IP) from 293 T cells transfected with Flag-OTUD5 plasmid and the lysates were subjected to SDS-PAGE and Coomassie Blue staining. B Enrichment analysis of mass spectrometry results showed that OTUD5 is involved in the mTOR signaling pathway. C IB analysis of whole-cell lysates (WCL) derived from 253 J and UM-UC-14 cells stably expressing shOTUD5 and shNC. shNC was used as the negative control. p-mTOR, t-mTOR, p-S6K, p-4EBP1 and OTUD5 were detected. ß-actin was used as the loading control. D IB analysis of WCL derived from T24 cells stably overexpressing OTUD5 and vector. Vector was used as the negative control. E, F UM-UC-14 shNC/shOTUD5 cells (E) and T24 Vec/oeOTUD5 cells (F) were subjected to amino acid starvation for 6 hours and resupplementation for 1 hour. p-S6K, p-4EBP1 and OTUD5 were detected. G, H UM-UC-14 shNC/shOTUD5 cells (G) and T24 Vec/oeOTUD5 cells (H) were treated with or without everolimus. I Representative images of OTUD5 and p-mTOR IHC staining in patient-derived bladder cancer tissues. Scale bars, left 500 µm, right 100 µm. J. Correlation analysis of OTUD5 and p-mTOR expression in patient-derived bladder cancer tissues. All data are presented as mean ± SD of 3 independent experiments.

Fig 5: OTUD5 promotes proliferation in bladder cancer.A, B, C EdU assay was performed to detect proliferation in 253 J and UM-UC-14 cells with OTUD5 knockdown and T24 cells with OTUD5 overexpression. **P < 0.01, ***P < 0.001. D Western blot was performed to determine the effect of OTUD5 on the expression of apoptosis markers (Bax, Cleaved-PARP, Cleaved-Caspase3) in bladder cancer cells. All data are presented as mean ± SD of three independent experiments. E The growth curves of xenografts in different groups. Tumor volumes were measured every three days from day 12 to day 30. ***P < 0.001. F Illustration of tumors excised from male nude mice in each group. G After 30 days, the nude mice were sacrificed, and UM-UC-14 cell xenografts were weighed. ***P < 0.001. H Immunohistochemical analysis of Ki67, Bax and Cleaved-PARP in tumor xenografts. I Western blot analysis of Bax, Cleaved-PARP and Cleaved-Caspase3 expression in tumor xenografts.