Figure 4

Effect of TGF-β1 on the expression of pAkt, ZO-1, Vimentin, Smurf2 and Smad7 in HPMCs, as assessed by Real-time PCR (Panels A-C), Cell Immuno- fluorescence (Panels D and E) and Western blot assay (Panels F, G & H1-H4). The bar graphs show a time-dependent mRNA expression of ZO-1 (Panel A), Vimentin (Panel B) and Smurf2 (Panel C) in HPMCs after treatment with TGF-β1 (5 ng/ml) for 0–72 hrs. Panels D and E: Confocal images of cells after staining with anti-pAkt, −Smurf2, −ZO-1 and -Vimentin antibodies. An increased nuclear expression of pAkt was observed in HPMCs following treatment with 5 ng/ml of TGF-β1 for 30 min (Panel D, upper panels). The Smurf2 expression in cytoplasm was also significantly increased (Panel D, lower panels). The ZO-1 expression was decreased in a time-dependent manner in HPMCs following treatment with TGF-β1 for 48 hr, which further decreased at 72 hr (Panel E, upper panels), In contrast, Vimentin expression was increased (Panel E, lower panels). Panel F & G: Western blot analyses of protein extract from HPMCs showed that pAkt and smurf2 expression initially increased in HPMCs, and then gradually decreased. While ZO-1 and Smad7 expression was gradually decreased over time. The Vimentin expression was significantly increased gradually over time. Panel H: The bar graphs represent the band density of Western blots. Values are the mean ± SEM, n = 3, * p <0.01 vs. control.