Figure 3

Effects of HG/SMG on cardiomyogenic differentiation of rBMSCs. rBMSCs were cultured under HG/SMG conditions with or without cardiomyocyte inducement medium for 1 or 3 days. RT-PCR analysis of β-MHC and GATA-4 mRNA expressions; (A) Electrophoresis graph of PCR products, (B) Gray intensity analysis of electrophoresis bands. (C) Western-blot detection of cTnT; CM, positive control, the RNA and protein were extracted from primary cardiomyocytes. Cont, cells were cultured under normal condition without inducer; C, rBMSCs were cultured with inducer for cardiomyocyte differentiation; H, HG culture; M, SMG culture; numbers 1, 3 indicate days of HG/SMG culture. Take samples: CH3, cells were cultured under HG conditions for 3 days with cardiomyogenic inducer; M3, the cells were cultured under SMG conditions for 3 days without inducer. SMG-/HG-, SMG/HG treated without inducer; SMG+/HG+, SMG/HG treated with inducer. * noted p < 0.05 and ** P < 0.01 compared with relative 0 d group (Cont, C) using student t-test analysis (n = 3). The experiments were conducted twice. The GAPDH house-keeping gene was used as a control. (D) immunocytochemistry analysis of cTnT in the cardiomyogenic differentiation of rBMSCs under HG/SMG conditions. a) The rBMSC group was used as a negative control. b) The rBMSCs treated with 5-aza only. c) The CH₃ group strongly expressed cTnT. d) The CM₃ group had few cTnT positive cells. Magnification × 200. HG increased the expression levels of GATA-4, β-MHC and cTnT in rBMSCs, whereas SMG decreased the expression levels.