Effect of BTXA on PTEN and caspase-3 expression, as well as PTEN methylation. (A) Effect of treatment of fibroblasts, which were cultured with or without 10 ng/ml of TGF-β1, with various concentrations (0.25, 0.5 and 1 UI/ml) of BTXA on the mRNA expression of PTEN, as determined by RT-qPCR. (B) Effects of BTXA on the protein levels of PTEN, pro-caspase-3 and cleaved-caspase-3, as detected by western blot analysis. (C) PTEN methylation was measured by methylation-specific PCR (MSP). M, methylated; U, unmethylated; MC, methylated control; UC, unmethylated control. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblast were treated with TGF-β1. Data are shown as the means ± SD, n=3. *P<0.05 and **P<0.01 vs. control without TGF-β1; ^P<0.05 and ^^P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1; PTEN, phosphatase and tensin homolog deleted on chromosome ten.