Figure 6.

Bay K8644 rescues osteogenic differentiation ability of Zmpste24−/− BMMSCs in vivo. (a) After intraperitoneal injection for 2 months, the protein expression levels of GSK3β, p‐GSK3β, β‐catenin, and active‐β‐catenin of BMMSCs from DMSO‐treated wild‐type, DMSO‐treated Zmpste24−/−, and Bay K8644‐treated Zmpste24−/− mice were confirmed by Western blot analysis (n = 6, 7, and 7). (b) After intraperitoneal injection for 2 months, Wnt target genes of cyclin D1 and c‐myc in BMMSCs from DMSO‐treated wild‐type, DMSO‐treated Zmpste24−/−, and Bay K8644‐treated Zmpste24−/− mice were explored by qRT–PCR (n = 6, 7, and 7). (c) After intraperitoneal injection for 2 months, expressions of osteogenic‐related genes of ALP, Runx2, and OCN in BMMSCs from DMSO‐treated wild‐type, DMSO‐treated Zmpste24−/−, and Bay K8644‐treated Zmpste24−/− mice were detected by qRT–PCR after osteogenic induction for 5 days (n = 6, 7, and 7). (d) After intraperitoneal injection for 2 months, expressions of osteogenic‐related proteins of ALP, Runx2, and OCN in BMMSCs from DMSO‐treated wild‐type, DMSO‐treated Zmpste24−/−, and Bay K8644‐treated Zmpste24−/− mice were detected by Western blot after osteogenic induction for 7 days (n = 6, 7, and 7). (e) After intraperitoneal injection for 2 months, alizarin red staining and quantification of BMMSCs from DMSO‐treated wild‐type, DMSO‐treated Zmpste24−/−, and Bay K8644‐treated Zmpste24−/− mice were performed to detect mineralized nodules after osteogenic induction for 14 days (n = 6, 7, and 7). (f) Schematic diagram shows that intraperitoneal injection of Bay K8644 improves defective osteogenic differentiation and ameliorates osteoporosis symptom through targeting Ca_v1.2 channel and canonical Wnt pathway of BMMSCs. 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 unpaired two‐tailed Student's t test