Figure 4.

Fibronectin regulates Ang II-stimulated AT1R protein expression through Integrin β1/Integrin-linked kinase signaling pathway. (A) Cardiac fibroblasts were transiently transfected with fibronectin siRNA (20 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. Integrin-β1 (ITGB1) protein expression was examined with β-actin as loading control. ** p < 0.01 (comparisons as depicted in the figure). (B) Cardiac fibroblasts were transiently transfected with ITGB1 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of Integrin-β1 silencing is also shown. ** p < 0.01 (comparisons as depicted in the Figure). (C) Cardiac fibroblasts were transiently transfected with ILK siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. ** p < 0.01 (comparisons as depicted in the Figure). (D) Cardiac fibroblasts were co-transfected with fibronectin siRNA (10 pmol) and ITGB1 cDNA overexpression plasmid (2 µg). Following transfection, the cells were revived in M199 with 10% serum for 12 h. Post-revival, the cells were serum-deprived for 24 h prior to treatment with Ang II for 12 h. Cells were collected and protein levels of AT1R, cIAP2, and collagen type 1 were examined, with β-actin as loading control. * p < 0.05 and ** p < 0.01 (comparisons as depicted in the Figure). Validation of fibronectin knockdown and ITGB1 overexpression is also shown. Data are representative of three independent experiments, n = 3, Mean ± SEM.