Figure 6.

Cfast associates with coactosin-like 1 (COTL1) protein to regulate the TGF-β signaling pathway

(A) Summary of distribution of proteins identified from cardiac fibroblasts using Cfast RNA pull-down coupled mass spectrometry. (B) Western blot of Cotl1 protein after lncRNA Cfast or control pull-down. Whole protein was used as positive control and poly(A) RNA used as negative control. (C) qRT-PCR of Cfast level after RNA immunoprecipitation using anti-COTL1 or control antibodies, in control or Cfast knockdown cardiac fibroblasts. Graph shows means ± SEM (n ≥ 3). p values were determined by two-way ANOVA (Sidak’s test). (D) Co-immunoprecipitation using anti-COTL1 or control IgG antibodies and western blot using anti-TRAP1 antibody, in control or Cfast knockdown cardiac fibroblasts. (E) Cardiac fibroblasts were infected with AD-COTL1 or control and COTL1 expression detected by qRT-PCR. Graph shows means ± SEM (n ≥ 5). p values were determined by Student’s t test. (F) qRT-PCR of the expression of indicated fibrotic genes from cardiac fibroblasts infected with AD-COTL1 or control. Graph shows means ± SEM (n ≥ 5). p values were determined by Student’s t test. (G) Cardiac fibroblasts were infected with AD-COTL1 or control, treated with TGF-β, and COTL1 expression detected by qRT-PCR. Graph shows means ± SEM (n ≥ 5). p values were determined by Student’s t test. (H) qRT-PCR of the expression of indicated fibrotic genes from cardiac fibroblasts infected with AD-COTL1 or control, treated with TGF-β. Graph shows means ± SEM (n ≥ 5). p values were determined by Student’s t test. (I) Gene set enrichment analysis of RNA-seq showed decreased TGF-β signaling in cardiac fibroblasts following Cfast knockdown.