Figure 4

VEGF-D knockdown promotes G1/S transition and cell proliferation but does not affect fibroblast activation. (A, B) MRC-5 cells were transfected with control siRNA (Ctrl) or VEGF-D siRNA for 48 h. Equal amounts of protein from whole cell lysates were analyzed by Western blotting with antibodies against VEGF-D, PCNA, α-SMA and β-actin (A). Ratio of PCNA to β-actin density was expressed as the fold-change relative to control siRNA alone (B). (C, D, E, F) Effects of siRNA-mediated VEGF-D silencing on cell cycle distribution of MRC-5 cells and NLFs. MRC-5 cells (C) and NLFs (E) were transfected with control siRNA or VEGF-D siRNA for 48 h. Cells cultured in DMEM containing 20% FBS were used as positive controls. Cells were collected, stained with propidium iodide and analyzed for DNA content. The number of cells in the G1, S and G2/M phases was calculated and expressed as a percentage of the total MRC-5 (D) and NLF (F) population. (G) Effects of siRNA-mediated VEGF-D silencing on the proliferative capacity of MRC-5 cells. MRC-5 cells were transfected with control siRNA (black) or VEGF-D siRNA (red). Cells cultured in DMEM containing 20% FBS were used as positive controls (blue). Cell proliferation was determined at the indicated time points after transfection. Data represent mean ± SEM of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (versus control siRNA); #P < 0.05; ^###P < 0.001 (versus VEGF-D siRNA), by the Student t test (B) and one-way ANOVA (D, FF G).