Fig. 2. NEDD4 potentiates malignant phenotypes of bladder cancer cells by promoting the stability and transcriptional activity of KLF8 through ubiquitination.

a IP assay of ubiquitination of KLF8 by NEDD4. b IP assay of ubiquitination of KLF8 by NEDD4 knockdown. c Analysis of KLF8 protein stability after CHX intervention. d Dual-luciferase assay to examine cyclin D1 promoter activity. e RT-qPCR and western blot assay to examine KLF8 silencing efficiency. f RT-qPCR to examine expressions of NEDD4 and KLF8 in response to oe-NEDD4 and siKLF8. g Western blot assay to examine the expression of NEDD4 and KLF8 normalized to GAPDH in response to oe-NEDD4 and siKLF8. h MTT assay to examine cell viability in response to oe-NEDD4 and siKLF8. i Flow cytometry analysis for apoptosis in response to oe-NEDD4 and siKLF8 under the induction of etoposide. j Transwell assay to examine cell migration in response to oe-NEDD4 and siKLF8 (scale bar: 50 μm). k Western blot assay to examine apoptosis-related proteins Bax and BCl-2, tumor suppressor proteins p53, p21, and metastasis-related proteins vimentin, N-cadherin, and E-cadherin normalized to GAPDH in response to oe-NEDD4 and siKLF8. *P < 0.05 versus the NC, oe-NC or oe-NC + si-NC group; ^#P < 0.05 versus the siKLF8 + oe-NC group. The experimental results are measurement data and are expressed as the mean ± standard deviation. Unpaired data with a normal distribution and homogeneity between two groups was compared using an unpaired t test. Comparisons among multiple groups were conducted by one-way ANOVA with Tukey’s post hoc test. Statistical analysis in relation to time-based measurements within each group was performed using repeated-measures ANOVA, followed by Bonferroni’s post hoc test. Cell experiments were independently repeated three times.