Figure 5

Effect of Akt-DN and LY294002 on ZO-1 and Vimentin expression in HPMCs. Panels A and B represent summary of Real-time RT-PCR. A reduced mRNA expression of ZO-1 (Panel A) and increased that of Vimentin (Panel B) in HPMCs stimulated with TGF-β1 was observed, while the expression was partially restored to pre-treatment levels with the PI3K inhibitor, LY294002 or by transfection of dominant-negative Akt (Akt-DN) plasmid. Panel C: By immunofluorescence microscopy, down-regulated ZO-1 and up-regulated Vimentin expression was observed in HPMCs subjected to TGF-β1 treatment, and it was normalized with transfection with Akt-DN or pretreatment with LY294002. On the other hand, TGF-β1 induced pAkt translocation to the nucleus in HPMCS, while partially blocked by treatment with either Akt-DN and LY294002. Similar results were also observed in nuclear extract from HPMCs, as detected by Western blot analyses (Panel D, D1-D2). Panel E: Western blot analyses showed down- regulated ZO-1 expression in HPMCs stimulated with TGF-β1, while partially reversed in that of treatment with LY294002 or Akt-DN plasmid. In contrast, an up-regulated Vimentin and pAkt expression was seen in HPMCs stimulated with TGF-β1 and partial normalization following the treatment with LY294002 or Akt-DN plasmid. Panels F, F1-F3: Bar graphs represent the density of relative bands of Western blots. Values are the mean ± SEM, n = 3, * p <0.01 vs. control, # p <0.01 vs.TGF-β1.