Fig. 5: SPRY4 functions via activating MEK- ERK1/2 pathways.

a Cell extracts were collected after knockdown or overexpression of SPRY4 in BM-MSCs without osteogenic induction. Then, western blot analysis was used to detect P-MEK, P-ERK, T-MEK, T-ERK, SPRY4 and RUNX2. b BM-MSCs were treated with ERK1/2 inhibitor U0126 at concentrations of 0, 10, 20 or 50 μM for 24 h to verify its efficiency. Western blot was performed to detect P-ERK, T-ERK, SPRY4 and RUNX2. c, d qRT-PCR (c) and western blot analysis (d) detected indicated osteogenic transcription factors and marker genes on day 6 of osteogenic differentiation after overexpressing SPRY4 and treatment with 10 or 50 μM U0126. Data were from three independent experiments using BM-MSCs derived from three healthy donors. e, f ALP staining and relative ALP activity assays were performed on day 6 of osteogenic differentiation after SPRY4 overexpression and treatment with 10 or 50 μM U0126. Data were from three independent experiments using BM-MSCs derived from 3 healthy donors. g, h Calcium deposition by Alizarin red S staining and quantification was performed on day 12 of osteogenic differentiation after SPRY4 overexpression and treatment with 10 or 50 μM U0126. Data were from three independent experiments using BM-MSCs derived from three healthy donors. GAPDH was used as a loading control in both qRT-PCR and western blot analyses. Data are shown as the means ± SD