Figure 6
From: Convergence of TGFβ and BMP signaling in regulating human bone marrow stromal cell differentiation
Downregulation of NOG promotes osteoblastic and adipocytic differentiation of hBMSC−Bone cells. (a) Quantification of cell viability of hBMSC−Bone cells transfected with NOG-siRNA scramble-siRNA (SCR) measured on days 2, 5, and 7. (b) qRT-PCR performed for NOG, TAGLN, ACTA2, TPM1, SMAD2, SMAD4 and SERPINB2 responsive genes in NOG-depleted vs. scramble-siRNA (SCR) transfected hBMSC−Bone cells under osteogenic induction conditions. The expression of each target gene was normalized to GAPDH. Data are shown as mean ± SD from three independent experiments, *p < 0.05; **p < 0.005, ***p < 0.0005. (c) OsteoImage™ staining (20× magnification) for hBMSC−Bone cells transfected with NOG or control siRNA under osteogenic induction conditions. The lower panel shows Alizarin Red S staining. The quantification of mineralized matrix formation for scramble-siRNA (SCR) and NOG-depleted cells is shown in the left panel, while the quantification of ALP activity under the same experimental conditions is shown in the right panel. Data are presented as relative mean mineralization ± SD from three independent experiments, n = 9; *p < 0.05, ***p < 0.0005. (d) qRT-PCR quantification of ALPL, RUNX2, OCN, and COL1A1osteogenic markers in scramble-siRNA (SCR) and NOG-depleted hBMSC−Bone cells exposed to osteogenic differentiation medium. The expression of each target gene was normalized to GAPDH. Data are presented as the means ± SD from three independent experiments, n = 9; *p < 0.05; **p < 0.005, ***p < 0.0005. (e) Nile red staining of hBMSC−Bone cells transfected with scramble-siRNA (SCR) or NOG-specific siRNA, which were then induced into adipocytes for 7 days (20× magnification). The cells were stained using oil red O staining as well. (lower panel, 20× magnification). The right panel shows the quantified fluorescence Nile red staining of mature oil-filled adipocytes. **p < 0.005. (f) qRT-PCR quantification for AN, LPL and PPARγ2 adiocytic markers. Expression of each target gene was normalized to GAPDH. Data are presented as the mean ± SD from three independent experiments, n = 9; *p < 0.05; **p < 0.005, ***p < 0.0005.
