Fig. 4

P311-induced EpMyT is mediated by TGFβ1/Smad signaling. a Western blot analysis showing expression of LAP-TGFβ1 and active TGFβ1 in primary mouse EpSCs. Relative densities of target proteins are also shown in comparison with the density of GAPDH. **P < 0.01. b Real-time PCR showing TGFβ1 mRNA levels in P311-transfected and control vector-transfected EpSCs. *P < 0.05. c Real-time PCR showing TβRI and TβRII mRNA levels in P311-transfected and control vector-transfected EpSCs. *P < 0.05 and **P < 0.01. d Phase-contrast microscopic observations of cell morphology 72 hours after the EpSCs were transfected with P311-containing vectors either with or without 5 μM LY2109761. e Immunofluorescence was performed to determine the expression of α-SMA (upper) and E-cadherin (lower) in EpSCs at 72 hours after transfection with P311 (left), the control vector (middle), and P311 with LY2109761 (right). α-SMA and E-cadherin were labeled using AF594 (red), and nuclei were stained using DAPI (blue). Scale bar, 25 μm. f Quantification of α-SMA⁺ and E-cadherin⁺ cells in an average of five regions. **P < 0.01 for P311 vs control vector; ##P < 0.01 for P311 vs P311 + LY2109761. g Western blot analysis was performed to determine α-SMA, vimentin, β₁-integrin, and E-cadherin levels. *P < 0.05 and **P < 0.01 for P311 vs control vector; and #P < 0.05 and ##P < 0.01 for P311 vs P311 + LY2109761. h Western blot analysis showing total and phosphorylated levels of Smad2 and Smad3 in primary EpSCs. LY2109761 was observed to have an inhibitory effect on Smad signals. *P < 0.05 and **P < 0.01 for P311 vs control vector; and ##P < 0.01 for P311 vs P311 + LY2109761. i Western blot analysis was performed to determine α-SMA, E-cadherin, and total and phosphorylated Smad3 levels after stimulation of Smad3 siRNA.*P < 0.05 and **P < 0.01 for P311 or P311 + mock siRNA vs the control vector; and ##P < 0.01 for P311 + Smad3 siRNA vs P311 or P311 + mock siRNA. j ELISA showing the level of total TGFβ1 in the culture supernatants of P311-transfected and control vector-transfected EpSCs. *P < 0.05. k Western blot analysis showing the expression of α-SMA in primary P311 WT and P311 KO EpSCs after the cells were incubated with or without TGFβ1 treatment. *P < 0.05 and **P < 0.01 relative to PBS control; ##P < 0.01 relative to P311 KO cells treated with TGFβ1. l Western blot analysis was performed to determine the total and phosphorylated Smad3 levels in P311 KO and P311 WT EpSCs with or without exogenous TGFβ1. Data shown as the mean ± SEM. **P < 0.01 relative to PBS control; #P < 0.05 relative to P311 KO cells treated with TGFβ1 (Color figure online)