Figure 3. KLF4 binds to the SM22α LacZ transgene in vitro and binding is attenuated by mutation of the G/C Repressor element after PDGF-BB or POVPC treatment in rat aortic smooth muscle cells.

A) Rat aortic smooth muscle cells were plated at 1×10^4. 24 hr later cells were transiently transfected using LT-Mirus with 300 ng of SM22α WT or G/C Repressor MT and increasing concentrations of pcDNA-KLF4. 24 hours following transfection, media was removed and replaced and 24 hours later cells were harvested and subjected to Bgal and protein assays. Results are the average of three independent experiments performed in triplicate. B&C) Rat aortic smooth muscle cells stably transfected with either SM22α or G/C Repressor mutant were plated at 1×10^4 and allowed to grow to confluency and then switched to serum free media for three days. Following serum starvation, cells were treated with 20 ng/ml PDGF-BB (B) or 5 ug/ml of POVPC (C) for 12 hours and then subject to ChIP analysis. * indicates significant binding as mentioned in materials and methods. D) Cells were plated and 24 hours following plating cells were treated with either si-control or si-KLF4. 24 hours following siRNA transfection, cells were treated with either vehicle or 20 ng/ml PDGF-BB for 12 hours. Cells were harvested and ChIP analysis was performed as described in part A. * indicates significant binding as mentioned in Fig 2D.