Figure 2: BIO inhibits cardiac fibroblast motility, negatively regulates pro-fibrotic factors, reduces Akt activity and increases p27 expression.

(Ai) Measurement of scratch area closure indicates that BIO treatment reduced neonatal rat cardiac fibroblast motility. n = 3, *p < 0.05, t-test. (Aii) Treatment with 5 μM BIO inhibited cardiac fibroblast motility. Untreated fibroblasts invaded the scratched area to a greater extent than BIO treated fibroblasts. In contrast, fibroblasts treated with 20 mM LiCl showed greater migration compared to untreated cardiac fibroblasts. Scale bar = 100 μm. n = 3, *p < 0.05, t-test. (Bi) BIO treatment for 3 days decreased mRNA expressions of the pro-fibrotic CCL11 and the anti-fibrotic IL-10 in 100 nM AngII-activated cardiac fibroblasts. Densitometry analyses were expressed as graphs. n = 3, *p < 0.05, t-test. (Bii) Quantitative PCR was performed to confirm the levels of CCL11 mRNA and IL-10 mRNA in AngII-activated cardiac fibroblasts. (Ci) BIO treatment for 3 days decreased mRNA expressions of the pro-fibrotic cytokines CTGF and TGF-β in cardiac fibroblasts. Densitometry analyses were expressed as graphs. n = 3, *p < 0.05, t-test. (Cii) Quantitative PCR was performed to confirm the levels of CTGF mRNA and TGF-β mRNA. *p < 0.05, t-test. (Di) BIO did not affect p21 mRNA expression and reduced p27 mRNA expression in cardiac fibroblasts. Cells were treated with BIO for 3 days. Densitometry analyses were expressed as graphs. n = 3, *p < 0.05, t-test. (Dii) Quantitative PCR was performed to confirm the levels of p21 mRNA and p27 mRNA in AngII-activated cardiac fibroblasts. *p < 0.05, t-test. (E) In BIO treatment of cardiac fibroblasts for 1 day, the protein levels of phosphorylated Akt, p21, and p27 were assessed. Densitometry analyses were expressed as graphs. n = 3, *p < 0.05, t-test. (F) Protein expressions of pAkt, p21 and p27 were determined by immunofluorescence staining in cardiac fibroblasts treated with BIO for 1 day. *p < 0.05, t-test. Scale bar = 100 μm.