Figure 4
The osteogenic role of T63 is dependent on RUNX2 expression. (a) Expression of RUNX2 mRNA and protein. MC3T3-E1 cells were treated with T63 for 48 h, and mRNA (left) and protein level (right) was determined by qRT-PCR and western blot, respectively. *p < 0.05 versus control (n = 3). (b) T63 increased the enrichment of RUNX2 on the promoter region of Alpl analyzed by ChIP assay. (c and d) Depletion of Runx2 suppressed T63-induced ALPL activity. MC3T3-E1 (c) or C3H10T1/2 (d) cells were transfected with Runx2 shRNA for 6 h, followed by T63 treatment for 6 days before subjected to ALPL assay. *p < 0.05, ***p < 0.001 versus drug free control. ## p < 0.01 versus respective control shRNA group (n = 3). Knockdown efficiency of Runx2 shRNAs was shown in the upper panels. Full-length blots and gels are shown in Supplementary Figs S5 and S6.
