Figure 4

Effects of Gremlin siRNA plasmid on cell proliferation and TGF-β1, CTGF and collagen IV expression in MMCs. MMCs were transfected with Gremlin siRNA (pGenesil1.1-shGrem1) or corresponding negative control (pGenesil1.1) plasmids, and incubated with HG (30 mM D-glucose) for 24 h. (A) Cell proliferation was examined using BrdU ELISA. Total (B) TGF-β1 and (C) CTGF protein levels were determined using western blot analysis. (D) Culture medium was collected for measurement of collagen IV concentration by radioimmunoassay. 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 pGenesil1.1 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 pGenesil1.1 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.