Figure 3.

Knockdown of FGFR2 induced by siRNA-inhibited osteogenic differentiation by decreasing Erk1/2 MAPK activation: (A) qRT-PCR assay showed mRNA FGFR2 expression after transfection with siRNA against FGFR2. (B) CCK-8 assays were carried out to assess cell proliferation. Knockdown of FGFR2 inhibited the proliferation of MC3T3-E1 cells. (C) Relative expressions of osteogenic marker measured by qRT-PCR and Western blot. The expression of Alp, ColIα2, Runx2, Opn and Ocn mRNA in differentiated MC3T3-E1 were remarkably decreased in siFGFR2 group. The level of ALP, COLI, RUNX2 protein were also decreased in siFGFR2 group. (D) ALP staining and quantitation of ALP activity showed that the ALP activity was decreased in the siFGFR2 group compared with the control. Alizarin red staining and quantitative test demonstrated there was less mineral content in the siFGFR2 group. (E) Western blot analysis demonstrated that the level of t-FGFR2, p-FGFR2 and p-Erk1/2 was decreased in the siFGFR2 group. There was no significant change in the expression of key proteins in other downstream pathways. The western blot results of FGF/FGFR2-Erk1/2 was circled by the red frame. p values were significant at ** p < 0.01, *** p < 0.001 and **** p < 0.0001.