FIGURE 3.

KLF11-mediated regulation of cPLA2α requires defined promoter site recognition and KLF11 binds to cPLA2α promoter in vivo.A, outlining of site-directed mutagenesis in GC-rich areas of the cPLA2α promoter reporter construct. B, compared with control, KLF11 was able to repress the cPLA2α-WT promoter but failed to repress the cPLA2α promoter that had cc to tt mutations in the distal GC-rich site (cPLA2α-WT (36.29 ± 17.7%) versus cPLA2α-SDM2 (80.8 ± 20.9%), p < 0.05). The mutations in the more proximal GC-rich site (SDM1) only partially relived the KLF11-dependent repression (52 ± 14.7%), which was not significantly different compared with cPLA2α-WT. C, electrophoretic mobility shift assay shows that binding of the KLF11-GST recombinant protein and digoxigenin-labeled fragment of the cPLA2α core promoter sequence was partially disrupted with SDM-1 mutations (lane 2) and completely disrupted by SDM-2 mutations (lane 3) in the GC-rich sequence of the cPLA2α promoter. Lane 1 represents the fragments containing the wild-type cPLA2α core promoter sequence (Wt-cPLA2α) and lanes 4–6 are negative controls. D, ChIP assay using FLO cell lysates shows that the cis-regulatory cPLA2α promoter sequence is enriched in immunoprecipitated samples from cells infected with KLF11-carrying adenovirus and absent in EV control-infected cells demonstrating that KLF11 can bind to the promoter of cPLA2α endogenously.