PTEN inhibits BMP2-induced osteogenic differentiation. C3H10T1/2 cells were cultured in BMP2-containing osteogenic medium for indicated periods. (A) ALP activity assay at day 3 (n=3). (B) ALP staining at day 7 (n=3). (C) qPCR analysis on day 3 (n=3). (D) qPCR for OC mRNA at day 5 (n=3). (E) Enzyme-linked immunosorbent assay for osteocalcin secretion in the supernatant at day 7 (n=3). (F) von Kossa staining in cells cultured in osteogenic medium for 2 weeks. The quantification of stains was performed using ImageJ (n=3). (G) Alizarin red staining in cells cultured in osteogenic medium for 4 weeks. The quantification of stains was performed using the ARed-Q assay. The sample solution was measured at OD 402 (n=3). Scale bar, 100 µm. *P<0.05; **P<0.01; ***P<0.001. BMP, bone morphogenetic protein; ALP, alkaline phosphatase; OC, osteocalcin; qPCR, quantitative PCR.