Fig. 5

The TGF-β1-driven mixed SMAD signaling inhibits osteogenesis, but not adipogenesis, of MSCs. A Western blots to evaluate the dose effects of SB on TGF-β1-induced pSMAD1/5/8 and pSMAD2/3 in C3H10T1/2 cells. Blots are representative of three independent experiments. B and C In vitro differentiation assays. C3H10T1/2 cells were primed with either low-dose or high-dose SB followed by treatment with TGF-β1 (1 nM) and/or BMP4 (1 nM) under adipogenic or osteogenic condition. (B) Oil red O staining (upper panel) and stained area quantification (low panel) were used to evaluate adipogenic capacity. C BCIP/NBT staining (upper panel) and intensity quantification (lower panel) were used to evaluate osteogenic capacity. n = 3 independent experiments. D In vivo adipogenesis assay. C3H10T1/2 cells primed with TGF-β1 (1 nM), BMP4 (1 nM) and/or SB (low or high dose) as indicated were used to perform ectopic adipocyte formation in mice. Cell plugs (yellow circle) were dissected, frozen-sectioned, and stained with Oil red O and hematoxylin. Scale bars, 100 μm. E-G Ex vivo osteogenesis assay. The foot bones from E16.5 mice were cultured in osteogenic medium supplemented with low-dose SB, TGF-β1 (1 nM) and/or BMP4 (1 nM). E The increased length of metatarsals was measured at indicated days. For more complete presentation, sample distributions in the TGF-β1 group (box a) and the TGF-β1 plus BMP4 group (box b) were placed independently on the right panels. Each point represented a single metatarsal. µCT images of the treated tibiae (F) were analyzed and a representative bone parameter (G) were shown. BV/TV: bone volume /total volume. n = 4 mice in each group. Data are means ± SD. For statistical analysis, ***P < 0.001 is compared to the control in the same group and determined by two-way ANOVA followed by multiple comparison test; ###P < 0.001 is determined by one-way ANOVA with multiple comparison test