Figure 7

Exogenous NOG suppresses osteoblastic and adipocytic differentiation of hBMSC^+Bone cells. (a) Quantification of cell viability measured on days 1, 4, and 6 for hBMSC^+Bone cells in the presence or absence of recombinant NOG (50 ng/ml). (b) qRT-PCR performed for TPM1, SMAD2 and SERPINB2 TGFβ responsive genes in hBMSC^+Bone cells in the presence or absence of recombinant NOG (10 ng/ml). The expression of each target gene was normalized to GAPDH. Data are presented as mean ± SD from three independent experiments, **p < 0.005. (c) OsteoImage™ staining (20× magnification) of hBMSC^+Bone cells which were induced into the osteoblast in the presence or absence of recombinant NOG. The lower panel shows Alizarin Red S staining. The quantification of mineralized matrix formation for vehicle or recombinant NOG-treated hBMSC^+Bone cells is shown (right panel). Data are presented as relative mean mineralization ± SD from three independent experiments, n = 9; *p < 0.0005. (d) qRT-PCR quantification of ALPL, RUNX2, OCN, and COL1A1 osteogenic markers in hBMSC^+Bone cells in the presence or absence of recombinant NOG (10 ng/ml) under osteogenic induction conditions. The expression of each target gene was normalized to GAPDH. Data are presented as the means ± SD from three independent experiments, n = 9; **p < 0.005, ***p < 0.0005. (e) hBMSC^+Bone cells were differentiated into adipocytes for 7 days under the indicated experimental conditions. Upper panel shows fluorescence Nile red staining of mature oil filled adipocytes (20× magnification), whilst the lower panel shows Oil red O staining for adipocytes (20× magnification). The lower panel shows the relative quantification of Nile red staining of mature oil-filled adipocytes. (f) qRT-PCR quantification for AP2, AN, LPL and PPARγ2 adipocytic markers. The expression of each target gene was normalized to GAPDH. Data are presented as mean ± SD from three independent experiments, n = 9; ***p < 0.0005.