Figure 2

Effects of expression plasmids on the expression of GFP and Gremlin in MMCs. Transient transfection of MMCs with Gremlin plasmid (pEGFP-N1-Grem1) and Gremlin siRNA plasmid (pGenesil1.1-shGrem1) induced GFP expression, indicating successful transfection. GFP expression was observed at 12, 24, and 48 h after transfection. MMCs were transfected with Gremlin plasmid (pEGFP-N1-Grem1), Gremlin siRNA plasmid (pGenesil1.1-shGrem1), or their corresponding negative control plasmids (pEGFP-N1or pGenesil1.1). They were then incubated with NG (5.5 mM D-glucose). At 48 h after transfection, total RNA and protein were extracted from the cells for Gremlin mRNA and protein expression. (A) GFP expression was observed through the comparative analysis of bright-field image and corresponding fluorescent image at 12, 24, and 48 h after Gremlin plasmid transfection (200×). (B) GFP expression was observed through the comparative analysis of bright field images and corresponding fluorescent images at 12, 24, and 48 h after Gremlin siRNA plasmid transfection (200×). (C) The expression of Gremlin mRNA was detected by real-time polymerase chain reaction (RT-PCR) analysis at 48 h after Gremlin plasmid transfection. (D) The expression of Gremlin mRNA was detected using RT-PCR analysis 48 h after Gremlin siRNA plasmid transfection. (E) The expression of Gremlin protein was analyzed using western blot analysis 48 h after Gremlin plasmid transfection. (F) The expression of Gremlin protein was analyzed using western blot analysis 48 h after Gremlin siRNA plasmid transfection. NG: MMCs 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; NG + V: MMCs transfected with pEGFP-N1 or pGenesil1.1 plasmid and incubated with 5.5 mM D-glucose. Values are expressed as means ± S.E.M. *P < 0.05, **P < 0.01 vs. NG.