Figure 3

TGF-β1 downregulates VEGF-D expression in MRC-5 cells via the JNK signaling pathway. (A, B, D, E, G, H, J, K) MRC-5 fibroblasts were pretreated for 1 h with wortmannin (100 nmol/L) (A, B), UO126 (10 µmol/L) (D, E), SB203580 (10 µmol/L) (G, H) or SP600125 (5 µmol/L) (J and K), specific chemical inhibitors for PI3K/Akt, MEK1/2, p38MAPK and JNK, respectively. Subsequently, cells were treated with TGF-β1 (5 ng/mL) for the indicated time. (A, D, G, J) MRC-5 fibroblasts were collected 1 h after TGF-β1 stimulation. Equal amounts of protein from whole cell lysates were analyzed by Western blotting with antibodies against phosphorylated and total Akt (A), phospho-p44/42 and total p44/42MAPK (D), phospho-HSP27 and total HSP27 (G), and phosphorylated and total c-Jun (J). (B, E, H, K) MRC-5 fibroblasts were collected 48 h after TGF-β1 stimulation. Equal amounts of protein from whole cell lysates were analyzed by Western blotting with antibodies against VEGF-D, α-SMA and β-actin. Ratio of VEGF-D to β-actin density was expressed as the fold-change relative to unstimulated control (C, F, I, L). Data represent meads ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, NS, 3ot significant, by one-way ANOVA.