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. 2010 Nov 26;12(1):56–62. doi: 10.1038/embor.2010.172

Figure 2.

Figure 2

KLF4 competes with KLF5 to bind to and regulate the miR-146a promoter. (A) 293A cells were transfected with pGL3-miR-146a-luc or pGL3-Basic. After 24 h, luciferase activities were measured. **P<0.01 compared with pGL3-Basic control. (B) Partial sequence of the miR-146a promoter and KLF4/KLF5-binding motifs. The underlined sequences are primers for ChIP assay. (C) Proteins and DNA in VSMCs were crosslinked. ChIP assays were performed with KLF4 or KLF5 antibodies and the miR-146a promoter region containing KLF4/KLF5-binding sites was amplified by PCR. (D) Recombinant KLF4 or KLF5 was incubated with a biotin-labelled probe containing KLF4/KLF5-binding sites in the miR-146a promoter. Protein–DNA complexes were separated by non-denaturing polyacrylamide gel electrophoresis and then visualized by chemiluminescence. Super-shift assays were performed by adding KLF4 or KLF5 antibodies. (E) Oligonucleotide pull-down assay was performed with biotin-labelled double-stranded oligonucleotides for the KLF4/KLF5-binding sites and their mutants as probes. The DNA-bound proteins were collected with streptavidin-agarose beads and analysed by western blotting with KLF4 or KLF5 antibodies. (F) 293A cells were co-transfected with pGL3-miR-146a-luc and KLF4 or KLF5 expression plasmid. After 24 h, luciferase activities were measured. *P<0.05 compared with empty vector pEGFP-N1 control. (G) 293A cells were co-transfected with a constant amount of PMT-KLF5 and with increasing amounts of pEGFP-KLF4, along with pGL3-miR-146a-luc. After 24 h, luciferase activities were measured. *P<0.05 compared with PMT-KLF5 alone. (H) 293A cells were co-transfected with a constant amount of pEGFP-KLF4 and with increasing amounts of PMT-KLF5, along with pGL3-miR-146a-luc. After 24 h, luciferase activities were measured. *P<0.05 compared with pEGFP-KLF4 alone. (I) VSMCs were transduced with Ad-KLF4, Ad-KLF5 or Ad-GFP. After 48 h, KLF4, KLF5 and miR-146a expression levels were detected by using western blotting and qRT–PCR. *P<0.05 compared with Ad-GFP control. All experiments were repeated three times. Ab, antibody; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; KLF, Krüppel-like factor; qRT–PCR, quantitative real-time PCR; VSMC, vascular smooth muscle cell.