Fig. 3

E3 ubiquitin ligase WWP1 promotes ubiquitination and degradation of KLF2 in BMSCs. a The protein level of KLF2 in BMSCs after treatment with CHX or CHX-MG132. *p < 0.05 vs. DMSO. ^#p < 0.05 vs. CHX. b The protein level of KLF2 after overexpression of ubiquitin ligases Smurf1, Smurf2, Itch, and WWP1 measured by Western blot analysis normalized to GAPDH. *p < 0.05 vs. oe-NC. c The protein level of KLF2 after overexpression of WWP1 and treatment with MG132 measured by Western blot analysis normalized to GAPDH. d The effects of Myc-labeled WWP1 on endogenous KLF2. The input was the positive control, and IgG was the negative control. e Exogenous KLF2 assessed by GST pull-down assay with GST-WWP1 fusion protein and KLF2 protein translated in vitro. The input was the positive control, and GST was the negative control. f Ubiquitination of KLF2 by adding ubiquitin UB after overexpression of WWP1. *p < 0.05 vs. oe-NC + DMSO. ^#p < 0.05 vs. oe-WWP1 + DMSO. g WWP1 protein level during osteogenic differentiation measured by Western blot analysis normalized to GAPDH. *p < 0.05 vs. PM. h Silencing efficiency of WWP1 evaluated by Western blot analysis normalized to GAPDH. i, j ALP activity of si-WWP1-transfected BMSCs (× 200). k The expression of osteogenic differentiation-related marker proteins (Runx2, OPN, and OCN) in si-WWP1-transfected BMSCs with osteogenic differentiation for 14 days evaluated by Western blot analysis normalized to GAPDH. *p < 0.05 vs. sh-NC. ^#p < 0.05 vs. hBMSCs treated with EV-inhibitor-NC. l The mineralization of BMSCs determined using ARS staining after treatment with si-WWP1 or si-NC. *p < 0.05 vs. BMSCs transfected with si-NC (× 200). The measurement data were shown as mean ± standard deviation. Data between the two groups were compared by independent sample t test. Comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post hoc test. Data comparison among the groups at different time points was analyzed by repeated-measures ANOVA, followed by Tukey’s post hoc test. The cell experiment was repeated three times