Figure 8.

Myadm knockdown/knockout increases the binding of KLF4 to the intergenic region of the p21/Cip1 promoter and the protein expression level without changing p21/Cip1 promoter activity. PASMCs isolated from Myadm^-/- rats or PASMCs transfected with siRNA against the Myadm gene were used for ChIP analysis together with ChIP-seq to map the genome-wide location of KLF4 binding region followed by Myadm knockdown or knockout, focusing on the p21/Cip2 promotor. A. Myadm knockdown/knockout increased KLF4 binding to the intergenic region of p21/Cip1 promoter. B, DNA-protein complexes were immunoprecipitated with anti-KLF4 Abs and were subjected to analysis using qRT-PCR followed by agarose gel electrophoresis showing PCR product. C, Growth-arrested PASMCs were co-transfected with KLF4-overexpressing plasmid (or control) and the firefly luciferase reporter gene containing p21 promoters and the transcription activities of p21 were measured according to the luciferase activity. D, KLF4 overexpression in PASMCs did not increase the mRNA expression level of p21/Cip1. E, PASMCs transfected with Ad-Myadm or Myamd-siRNA were subjected to cross co-immunoprecipitation (IP) and Western blotting (IB) to detect KLF4-SMAD4 complex formation. In the left panel, cell lysates were immunoprecipitated with anti-KLF4 resin and then immunoblotted with KLF4 and SMAD4 antibodies. In the right panel, cell lysates were immunoprecipitated with anti-SMAD4 resin and then immunoblotted with SMAD4 and KLF4 antibodies. For C-D, n=6-8 in each group.