Figure 4

Downregulation of SERPINB2 promotes osteoblastic and adipocytic differentiation of hBMSC^−Bone cells. (a) Alamarblue quantification for cell viability of hBMSC^−Bone cells transfected with scramble-siRNA (SCR) or SERPINB2-siRNA on days 2 and 4. (b) qRT-PCR for SERPINB2, TAGLN, ACTA2, TPM1, COL1A2, SMAD2 and SMAD4 TGFβ responsive genes in SERPINB2-depleted vs. scramble-siRNA (SCR) control cells. The expression of each target gene was normalized to GAPDH. Data are presented as mean ± SD from three independent experiments; **p < 0.005, ***p < 0.0005. (c) Shows the OsteoImage™ staining (20× magnification) in differentiated hBMSC^−Bone cells post SERPINB2 knockdown compared to scramble-siRNA transfected control cells. The lower panel shows Alizarin Red S staining. Quantification of mineralized matrix formation under different treatments is shown on the right panel. Data are presented as mean mineralization ± SD from three independent experiments, n = 9; ***p < 0.0005. (d) qRT-PCR quantification of ALPL, RUNX2, OCN, OPN, BMP4, and COL1A1 osteogenic markers mRNA expression in SERPINB2-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 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 mature oil filled adipocytes (20× magnification) in hBMSC^−Bone cells on day 7 post adipocytic differentiation. Oil red O staining is shown in the lower panel (20× magnification). The right panel shows quantification of Nile red staining, ***p < 0.0005. (f) qRT-PCR quantification for aP2, LPL and PPARG2 adipogenic markers. Expression of each target gene was normalized to GAPDH. Data are presented as means ± SD from three independent experiments, n = 9; *p < 0.05; **p < 0.005.