Figure 1.

KLF4 up-regulates mfn-2 expression in VSMCs. (A) VSMCs were serum-starved for 24 h and then treated with ATRA (10 μmol/L) for different times (12, 24, 48, and 72 h). Cells were collected and Western blotting was performed using anti-KLF4 and anti-mfn-2 antibodies to examine the expression of KLF4 and mfn-2, respectively. β-Actin was used as a control for equal protein loading. (B) KLF4 and mfn-2 expression induced by ATRA was detected by quantitative real-time RT-PCR. VSMCs were treated with ATRA for different times (6, 12, 24, and 48 h), and total RNA was isolated from VSMCs and subjected to quantitative real-time RT-PCR analysis by using specific primers for KLF4 and mfn-2. GAPDH was used as an internal control. The bars represent the means±SEM from three independent experiments. ^bP<0.05 vs control (0 h of ATRA). (C) Adenovirus-mediated KLF4 overexpression in VSMCs. VSMCs were infected with pAd-KLF4 or pAd for 24 h before harvesting. Western blotting was performed using antibodies against KLF4 or β-actin (as a loading control). (D) VSMCs were infected with pAd-KLF4 for the indicated times. The cell lysates were subjected to Western blotting with antibodies against mfn-2 and β-actin. (E) VSMCs were infected with pAd-KLF4 for the indicated times. Total RNA was isolated from VSMCs and subjected to quantitative real-time RT-PCR analysis by using specific primers for mfn-2. GAPDH was used as an internal control. The bars represent the means±SEM. from three independent experiments. ^bP<0.05 vs control (0 h of pAd-KLF4). (F) The effects of KLF4 knockdown of by siRNA on ATRA-induced mfn-2 gene expression. VSMCs were transfected with KLF4-specific siRNA (KLF4-siRNA) or non-silencing control siRNA (NS-siRNA) for 24 h. Cells were treated with ATRA (10 μmol/L) for 24 h, and then harvested and subjected to Western blotting to determine the levels of KLF4 and mfn-2. β-Actin was used as a control for equal protein loading.