Figure 2 of Doudnikoff, Mol Vis 2024; 30:160-166.


Figure 2. TGF-β pathway activation in UM cell lines. A: Western blot analysis of SMAD2, SMAD3, and phospho-SMAD2/3 expressions in response to TGF-β treatment (1 ng/ml TGF-β, time course experiment from 2 to 24 h of treatment) in the Mel270 cell line, using β-actin as a loading control in all western blot experiments (n = 1 biological replicate). B and C: Western blot analysis of SMAD2, SMAD3, and phospho-SMAD2/3 expressions in response to TGF-β, Ly (LY2157299, type 1 TGF-β receptor inhibitor), or a combined treatment (1 ng/mL TGF-β and 10 mM LY2157299; 1 h; n = 3 biological replicates). D: Quantification of phospho-SMAD2/SMAD2 and phospho-SMAD3/SMAD3 Mel270 western blot signal intensities relative to (C) (n = 3 biological replicates). E: Expression levels of 2 well-known TGF-β target genes (SERPINE1 and SMAD7) in UM cell lines in response to TGF-β, LY2157299 (type 1 TGF-β receptor inhibitor) or a combined treatment (1 ng/ml TGF-β and 10 mM LY2157299; 16 h), evaluated using RT-qPCR (n = 3 biological replicates). F: Cell viability evaluated using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay in UM cell lines in response to TGF-β, LY2157299, or a combined treatment (1 ng/ml TGF-β and 10 mM LY2157299; 16 h) from days 1 to 4 after treatment. When cells were incubated for more than 2 days in culture, the same treatment was reapplied on day 2 (n = 3 biological replicates). Statistical analyses for (D–F) were performed using a t test. Data are presented as mean ± SD from three independent biological replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. TGFβ, transforming growth factor β; Ly, LY2157299; P-SMAD2, phospho-SMAD2; P-SMAD3, phospho-SMAD3; BSA, BSA, DMSO, dimethylsulfoxyde.