Figure 3

CM-Adipo inhibits BMP2-induced osteoblast differentiation of mBMSCs. a Studying the effect of CM-Adipo versus CM-Control on different osteogenic signaling pathways. Cultured mBMSCs were induced for osteogenesis without (control) or with regular osteogenic induction medium (induced), PDGF-BB (100 ng/ml), Wnt3a (10 ng/ml), BMP2 (100 ng/ml) and insulin (10ug/ml) in 100% of either CM-Adipo or CM-Control. ALP activity was quantified after 6 days of induction and represented as fold change over control non-induced cells. b Dose-dependent inhibitory effect of CM-Adipo on BMP2-induced matrix mineralization in m BMSCs. Alizarine Red staining and its quantification were performed after 12 days of induction. c qPCR analysis of osteoblastic gene expression in mBMSCs induced to osteoblast differentiation by BMP2 in either CM-Adipo or CM-Control for 6 days. d Western blot analysis of Smad1/5/8 phosphorylation in BMP2 treated mBMSCs in either CM-Adipo or CM-Control for 5-20 min. Values are mean ± SD of three independent experiments, (*p < 0.05, **p < 0.005)