Figure 2.

TGF-β1 increases PTBP3 protein levels of LUAD cell lines in a Smad-dependent manner. (a) Western-blot analysis of PTBP3 and EMT-related genes in four LUAD cell lines treated by TGF-β1 (5 ng/ml or 10 ng/ml) as indicated days. “d” represents the days for LUAD cell lines treated by TGF-β1. (b) Western-blot analysis of PTBP3, Smad4, N-cadherin and E-cadherin expression in A549 cells treated with control or Smad4 siRNA (100 nM) for 24 h and then treated by TGF-β1 (5 ng/ml) for another 24 h. (c) Western-blot analysis of PTBP3, N-cadherin and E-cadherin expression in A549 cells treated with 5 ng/ml TGF-β1 and 0–10 μM TGF-β type I receptor inhibitor SB431542 for 24 h. (d) A schematic diagram of the PTBP3 promoter cloned into pGL3 vector. Two predicted SBEs within the PTBP3 promoter are shown and PTBP3 promoter constructs containing mutations in these two SBEs are generated. The mutant bases of two predicted SBEs are shown in red bold letters. (e) A549 cells were co-transfected with pRL-TK plasmid and indicated constructs for 24 h, and then treated with or without TGF-β1 (5 ng/ml) for another 24 h. Then the cells were lysed to analyze luciferase activity. pRL-TK plasmid was used for internal control. (f) ChIP assay for p-Smad3 binding to two SBEs in the promoter of PTBP3. A549 cells left untreated or treated with 5 ng/ml TGF-β1 for 6 h were harvested and subjected to ChIP with isogenic IgG or anti-p-Smad3 antibody. The enrichment of the precipitated DNA by p-Smad3 antibody versus the IgG was analyzed by qPCR with the specific primers for SBE1 and SBE2. Data were presented as percent input value. (g) A schematic diagram shows that exogenous TGF-β1 induces transcription of PTBP3 in a Smad-dependent manner. GAPDH was used as an internal control for western-blot analysis. Error bars are mean ± SD from three independent experiments and Student’s t-test was used to analyze differences between groups. **P < 0.05, **P < 0.01, ***P < 0.001.