Figure 5

Effects of Gremlin on pERK1/2 protein expression in MMCs. MMCs were cultured in NG then stimulated (M) or HG for 6, 12, 24, and 48 h. At the end of each time point, total cell protein contents were extracted for assessment of pERK1/2 protein expression. MMCs were transfected with Gremlin (pEGFP-N1-Grem1), Gremlin siRNA (pGenesil1.1-shGrem1) or their corresponding negative control (pEGFP-N1 or pGenesil1.1, respectively) plasmids, and incubated with HG for 24 h. Protein was then extracted from the cells for evaluation of pERK1/2 protein expression. (A) The level of pERK1/2 protein expression was assessed using western blot analysis in MMCs incubated with HG for the indicated times (0–48 h). Values are expressed as means ± S.E.M. *P < 0.05, **P < 0.01 vs. NG. (B) MMCs were transfected with Gremlin or the corresponding negative control plasmids and the expression of pERK1/2 protein was assessed by western blot analysis 24 h after HG stimulation. (C) MMCs were transfected with Gremlin siRNA or the corresponding negative control plasmids and the expression of pERK1/2 protein was assessed using western blot analysis 24 h after HG stimulation. NG: MMCs incubated with 5.5 mM D-glucose; NG + M: MMCs incubated with 5.5 mM D-glucose plus 24.5 mM mannitol; NG + V: MMCs transfected with pEGFP-N1/pGenesil1.1 plasmids and incubated with 5.5 mM D-glucose; NG + P: MMCs transfected with pEGFP-N1-Grem1 plasmid and incubated with 5.5 mM D-glucose; NG + gremlin si: MMCs transfected with pGenesil1.1-shGrem1 plasmid and incubated with 5.5 mM D-glucose; HG: MMCs incubated with 30 mM D-glucose; HG + V: MMCs transfected with pEGFP-N1/pGenesil1.1 plasmids and incubated with 30 mM D-glucose; HG + P: MMCs transfected with pEGFP-N1-Grem1 plasmid and incubated with 30 mM D-glucose; HG + gremlin si: MMCs transfected with pGenesil1.1-shGrem1 plasmid and incubated with 30 mM D-glucose. Values are expressed as means ± S.E.M. *P < 0.05,**P < 0.01, vs. NG, ^# P < 0.05,^## P < 0.01, vs. HG.