FIGURE 1.

RARα mediated ATRA-induced Klf4 expression. A, VSMCs were treated with 10 μm of ATRA for 0, 6, 12, or 24 h. Crude proteins were extracted from the treated cells and then subjected to Western blot with antibodies against KLF4, RARα, RARβ, or RARγ. β-Actin was used as a control for equal protein loading. Left, blots from a representative experiment. Right, densitometry; results were normalized to β-actin. The bars represent the means ± S.E. from three independent experiments. *, p < 0.05 versus ATRA-free group. B, VSMCs were transfected with si-RARα, si-RARβ, si-RARγ, or si-NS for 24 h and then treated with 10 μm of ATRA for 24 h. The cell lysates were analyzed by Western blot with antibodies against KLF4, RARα, RARβ, or RARγ. β-Actin was the loading control. C, VSMCs were transfected with si-RARα, si-RARβ, si-RARγ, or si-NS for 24 h and then treated with 10 μm of ATRA for 24 h. Total RNA was isolated and subjected to quantitative RT-PCR. The bars represent the means ± S.E. from three independent experiments. *, p < 0.05 versus si-NS-treated and ATRA-free group (first bar). D, VSMCs were pretreated with 20 μm of Ro 41-5253 for 2 h prior to exposure to ATRA (10 μm) for 24 h. Crude proteins from cell lysates were analyzed by Western blot with antibodies against KLF4 and RARα. β-Actin was the loading control. E, VSMCs were infected with Ad-null or Ad-GFP-RARα for 24 h prior to the exposure to ATRA (10 μm) for 24 h. Crude proteins from cell lysates were analyzed by Western blot with antibodies against KLF4 or RARα. β-Actin was the loading control. F, CHO-K1 cells were co-transfected with a Klf4 promoter-reporter construct and RARα, RARβ, or RARγ expression plasmid (GFP-RARα, GFP-RARβ, or GFP-RARγ) for 24 h and then treated with 10 μm of ATRA for 24 h. Cell lysates were subjected to luciferase activity assays using the dual luciferase reporter assay system, and the luciferase activity was normalized to pRL-TK activity. The bars represent the means ± S.E. from three independent experiments. * and #, p < 0.05 versus the respective control group. Expression level of GFP, GFP-RARα, GFP-RARβ, and GFP-RARγ was assessed by Western blot analysis. β-Actin was used as a control for equal protein loading. G, CHO-K1 cells were pretreated with Ro 41-5253, LE135, or MM11253 (antagonists of RARα, RARβ, or RARγ, respectively) for 2 h and then stimulated with 10 μm of ATRA for 24 h. Luciferase activity of the Klf4 promoter-reporter construct was measured as described above. *, p < 0.05 versus ATRA-treated group (second bar).