Fig. 7.

Wnt/β-catenin signaling induces cultured CF activation and ECM gene expression downstream of Tgfβ signaling in vitro. a Representative images of αSMA (green) staining in cultured P60 CFs under indicated conditions are shown. Scale bar = 100 μm. b Wnt1 (100 ng/ml) and TGFβ1 (10 ng/ml) induces significantly more αSMA⁺ CFs relative to DMSO controls in cultured P60 CFs. Inhibition of Wnt/β-catenin signaling by XAV939 (5 μM) abrogated Wnt1-induced αSMA expression in CF and also partially suppressed TGFβ1-induced αSMA expression. *P < 0.05 vs. DMSO groups, ^† P < 0.05 vs. Control. c mRNA expression of the Wnt target gene Axin2, and ECM genes Col1a1, Col3a1, Postn, and Acta2 in P60 CFs with indicated treatments was measured by quantitative PCR. *P < 0.05 vs. control, ^† P < 0.05 vs. DMSO. N = 4 independent cultures per group. Statistical significance was determined by Kruskal–Wallis tests followed by Mann–Whitney U tests for pairwise comparisons using Bonferonni adjustments to control for multiple testing. d ChIP assays were performed using P0 cultured CFs treated with BIO (2 μM) to determine the binding of β-catenin in protein complexes with TCF/LEF consensus containing proximal sequences of Axin2, Col3a1, and Postn genes. Compared to IgG negative control, binding of β-catenin to Col3a1 and Postn was significantly enriched. Axin2 was used as positive control. N = 4 independent cultures per group. Fold enrichment of DNA binding in ChIP assays was analyzed using one sample Wilcoxon signed-rank test compared to one. Data points are shown with median and interquartile ranges indicated. *P < 0.05 vs. IgG control