Fig 2: Effects of TGF-β receptor inhibition on EMT markers in nickel-treated cells. (a) BEAS-2B (1 × 106 cells/6 cm dish) and (b) A549 (5 × 105 cells/6 cm dish) cells were treated with NiCl2 (0.5 and 1 mM, respectively) or TGF-β (10 ng/ml) with or without SB525334 (10 μM) for 72 h and the protein levels were analyzed on Western blot. β-actin was used as the internal control. The relative ratios of Fibronectin/β-actin, E-cadherin/β-actin, HIF-1α/β-actin, TAB2/β-actin, ENOSF1/β-actin, and RAB6A/β-actin are shown. (c) BEAS-2B cells (1 × 106 cells/6 cm dish) were treated with NiCl2 (0.5 mM) for 15 min, 30 min, 1 h and 24 h or TGF-β (10 ng/ml, positive control) for 30 min. The protein levels were analyzed on Western blot. β-actin was used as the internal control. The relative ratios of p-Smad2/Smad2, p-Smad3/Smad3 and Smad4/β-actin are shown.

Fig 3: TF cytoplasmic domain phosphorylation–dependent increased TGF-β1 activation in clinical setting of MI.(A) Representative confocal images of phosphorylation status of TF in infarcted myocardium obtained from WT or TFΔCT mice after 7 days. Representative images and quantification of biological replicates. Kruskal-Wallis test and Dunn’s multiple-comparison test; n = 3–4 animals per group. Scale bars: 50 μm. (B) Representative immunofluorescence confocal microscopy images of CD45+ and CD45/p-TF double-positive cells in human myocardium specimens obtained from n = 5 nonischemic (NI) donor hearts and n = 7 IHF patients. Quantification of biological replicates. Mann-Whitney test. Scale bars: 50 μm. (C and D) Western blot analysis and quantification of human LV tissue obtained from n = 5 nonischemic donor hearts and n = 9 IHF patients for p-TF (normalized to total TF) and TF (C) or TGF-β1 (normalized to GAPDH) and p-SMAD2 (normalized to total SMAD2) (D). Mann-Whitney test. Data are shown as mean ± SEM. *P < 0.05.

Fig 4: 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.

Fig 5: Chondrogenic differentiation of human BMSCs lacking CRLF1 or overexpressing CRLF1. (A) Alcian blue staining of sections of BMSC pellets transfected with control siRNA (siCon) or siRNA specific for CRLF1 (siCRLF1) after 6 days in chondrogenic differentiation medium; sections of BMSC pellet transduced with empty AAV (AAV) or transduced with AAV containing CRLF1 (AAV-CRLF1) after 6 days in chondrogenic differentiation medium. Bar, 100 µm. (B) Phosphorylated (p-Smad) and total Smad2 and Smad3 immunoblots of lysates from BMSCs transduced with empty AAV (AAV) or from BMSCs transduced with AAV-CRLF1. Cells were treated for 60 min with transforming growth factor beta 1 (TGFβ1).