FIGURE 3.
TGF-β-induced α-SMA induction and transcription in RLE-6TN cells is Smad-dependent. Representative Western blot (A) and quantitative analysis (B) of α-SMA protein in RLE-6TN cells treated with TGF-β1 (or vehicle DMSO) ± SIS3. M denotes medium. Lamin A/C is used as a loading control (n = 3, *, p < 0.05 (significantly different from DMSO). Western blot (C) and quantitative analysis of Smad3 (D) and α-SMA (E) protein using cell lysate from RLE-6TN cells transduced with lentivirus expressing shRNA for Smad3 or control pGIPZ nonsilencing shRNA followed by TGF-β1 treatment. Lamin A/C and GAPDH were used as loading controls (n = 3, *, p < 0.05, significantly different from control shRNA). F, RLE-6TN cells were transfected with 764-bp α-SMA reporter, followed by treatment with TGF-β1 (2.5 ng/ml) for 24 h in the presence and absence of SIS3 (6 μm). Luciferase activity was normalized to Renilla luciferase activity (n = 5, *, p < 0.05 compared with TGF-β1 vehicle and TGF-β1/SIS3 together). G, co-transfection of RLE-6TN cells with rat α-SMA promoter reporter, together with Smad3 expression plasmid pRK-5F/Smad3 or empty vector pRK-5F, followed by TGF-β1 treatment for 48 h. Firefly luciferase activity is normalized to Renilla luciferase activity (n = 3, *, p < 0.05, significantly different from the pRK-5F). H, co-transfection of RLE-6TN cells with wild type α-SMAp-Luc and SBE1 and SBE2 mutants α-SMAp-Luc-SBEm1 and α-SMAp-Luc-SBEm2, followed by TGF-β1 treatment for 15 h. Firefly luciferase activity is normalized to Renilla luciferase activity (n = 8, *, p < 0.05; NS, not significantly different).