Fig. 4: USP53 regulates osteogenic differentiation in hBMSCs through Wnt/β-catenin signaling.

a, b Expression levels of Wnt/β-catenin signaling pathway mediators were investigated by immunoblotting in USP53-overexpressing (a) or -knockdown (b) hBMSCs in osteogenic medium for 7 days. Data were quantified using ImageJ. c Immunoblot analysis of Wnt3a-induced osteogenic marker expression. Data were quantified using ImageJ. d Immunoblot analysis of DKK1-induced osteogenic marker expression. Data were quantified using ImageJ. e Immunoblot analysis of β-catenin-linked polyubiquitination with overexpression of USP53. β-Catenin ubiquitination (top panel) and protein expression assays (bottom panel) were evaluated. f Immunoblot analysis of β-catenin-linked polyubiquitination with siUSP53. β-Catenin ubiquitination (top panel) and protein expression assays (bottom panel) were evaluated. g hBMSCs were transfected with Flag-β-catenin overexpression plasmid and control or Flag-USP53 and then treated with cycloheximide (100 μg/mL) and Wnt3a. Immunoblots with active β-catenin protein at the indicated time points. n = 3.