Figure 4.
Bay K8644 treatment activates Cav1.2 and rescues the impaired osteogenic differentiation of Zmpste24−/− BMMSCs. (a) After treated by DMSO or 10‐6, 10‐7, and 10‐8 M Bay K8644 for 3 days, the proliferation of Zmpste24−/− BMMSCs was detected by MTT (n = 3). (b) After treated by DMSO or 10‐6, 10‐7, and 10‐8 M Bay K8644 for 3 days, intracellular calcium current of Zmpste24−/− BMMSCs was explored by laser confocal microscopy (n = 3). (c) After treated by DMSO or 10‐6, 10‐7, and 10‐8 M Bay K8644 for 3 days, intracellular calcium concentration of Zmpste24−/− BMMSCs was explored by flow cytometry (n = 3). (d) After treated by 10‐7 M Bay K8644 or DMSO for 3 days, the protein expression levels of GSK3β, p‐GSK3β, β‐catenin, and active‐β‐catenin in Zmpste24−/− BMMSCs were confirmed by Western blot analysis (n = 3). (e) After treated by 10‐7 M Bay K8644 or DMSO for 3 days, Wnt target genes of cyclin D1 and c‐myc were explored by qRT–PCR (n = 3). (f) 48 hr after stimulated by DMSO or Bay K8644 in progerin‐overexpressed 293T cells, TOP/FOP, and Renilla luciferase plasmid were transfected, and TOP/FOP reporter assay was performed to detect the activity of β‐catenin after 24 hr (n = 3). (g) After osteogenic induction for 7 days, expressions of osteogenic‐related proteins of ALP, Runx2, and OCN in Zmpste24−/− BMMSCs of DMSO‐treated, Bay K8644‐treated, and Bay K8644‐treated in the context of Cav1.2 siRNA were explored (n = 3). (h) After osteogenic induction for 14 days, mineralized nodule formations in Zmpste24−/− BMMSCs of DMSO‐treated, Bay K8644‐treated and Bay K8644‐treated in the context of Cav1.2 siRNA were explored (n = 3). The expression levels of the target genes and proteins were normalized to GAPDH. Scale bar, 50 um. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, which was determined by paired two‐tailed Student's t test