Fig 1: 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. ß-actin was used as the loading control. n = 6 mice per treatment group, data are presented as mean ± SD.
Fig 2: OTUD5 stabilizes RNF186 by deubiquitination, leading to sestrin2 degradation.A Protein binding prediction chart for OTUD5 and RNF186. B Confocal immunofluorescence microscopic analysis of OTUD5 and RNF186 in 253 J and UM-UC-14 cells. Scale bars represent 10 µm. C IB analysis of WCL and anti-HA immunoprecipitates (IPs) derived from 293 T cells transfected with Flag-OTUD5 and HA-tagged RNF186, pcDNA3.1 was used as the control. D, E OTUD5 interacts with RNF186. Co-immunoprecipitation (co-IP) of OTUD5 and RNF186 was assayed in 253 J and UM-UC-14 cells. Immunoprecipitation (IP) was performed using the antibody against OTUD5, and the endogenous interaction between OTUD5 and RNF186 was determined by Western blotting using the antibody against RNF186. F IB analysis of WCL derived from 253 J and UM-UC-14 cells stably expressing shOTUD5 and T24 cells stably overexpressing OTUD5. RNF186 and sestrin2 were detected, ß-actin was used as the loading control. G GST pull-down assay revealed the direct interaction between RNF186 and OTUD5. The upper panel presents the result of IB using the antibody against HA, and the lower panels show Coomassie blue staining of the purified proteins. H IB analysis of WCL and Ni-NTA pull-down products derived from 293 T cells transfected with Flag-OTUD5 WT, Flag-OTUD5 C224S, HA-RNF186 and His-Ub. 20 µM MG132 was added 6 h before harvesting the cells. I OTUD5 knockdown cells (shOTUD5) as well as parental UM-UC-14 cells (shNC) were treated with 100 µg/ml cycloheximide (CHX) for the indicated time period before harvesting. Equal amounts of WCL were immunoblotted with the indicated antibodies. J Quantification of the band intensities in (I). RNF186 levels were normalized to the corresponding ß-actin levels, then normalized to the t = 0 h RNF186 level. K IB analysis of WCL derived from 293 T cells transfected with HA-RNF186, Flag-EV and Flag-OTUD5. Cells were treated with 100 µg/ml CHX for the indicated time period before harvesting. EV empty vector. L Quantification of the band intensities in K. RNF186 levels were normalized to the corresponding ß-actin levels, then normalized to the t = 0 h RNF186 level. All data are presented as mean ± SD of 3 independent experiments.
Fig 3: Schematic models.Upper: OTUD5 exerts its functions through the OTUD5-RNF186-sestrin2-mTOR axis to promote bladder cancer proliferation. Bottom: Downregulation of OTUD5 and everolimus show inhibitory effects on bladder cancer growth.
Supplier Page from Thermo Fisher Scientific for RNF186 Antibody