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. 2013 Sep;183(3):964–974. doi: 10.1016/j.ajpath.2013.05.011

Figure 3.

Figure 3

In vitro analysis of lentivector-delivered shRNA transduction and VEGFA knockdown in rMC-1 cells, and VEGFA signaling in a co-culture model of rMC-1s and hRMVECs. A: Diagram of the pFmCD44.1GW lentivector plasmid containing the glia-specific CD44 promoter driving an miR-30–based shRNA cassette and a GFP marker. B: GFP expression in rMC-1s and hRMVECs. Real-time PCR of VEGFA mRNA in rMC-1s (C) and ELISA of VEGFA protein in culture media of rMC-1s transduced with lentivirus (D). E and F: Knockdown of VEGFA in rMC-1s by lentivector-VEGFA-shRNA reduces co-culture–induced p-VEGFR2 in hRMVECs. Representative gels of p-VEGFR2 in hRMVECs grown in contact with lentivector VEGFA-shRNA or luciferase-shRNA–transduced rMC-1s (E) (solo cultured, treated with VEGFA, and grown in contact with rMC-1s) and in hRMVECs grown in contact with lentivector VEGFA-shRNA– or luciferase-shRNA–transduced rMC-1s (F). *P < 0.05, ∗∗∗P < 0.001 versus uninfected; †P < 0.05, †††P < 0.001 versus luciferase-shRNA. Data shown in C and D are representative of six independent samples. Results are means ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.