FIGURE 5.

MKL1 interacts with NF-κB to activate miR-155 transcription in macrophages. (A) A miR-155 promoter-luciferase construct was transfected into HEK293 cells with or without MKL1. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (B) miR-155 promoter-luciferase constructs of various lengths were transfected into HEK293 cells with or without MKL1. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (C) Wild type or NF-κB site mutant miR-155 promoter-luciferase construct was transfected into HEK293 cells with or without MKL1. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (D) RAW cells were treated with endothelin and harvested at indicated time points. ChIP assays were performed with anti-MKL1. (E) RAW cells were treated with or without endothelin for 24h. Re-ChIP assays were performed with indicated antibodies. (F,G) RAW cells were transfected with siRNA targeting NF-κB or SCR followed by treatment with endothelin. Knockdown efficiencies and were verified by Western. ChIP assays were performed with anti-MKL1. (H) RAW cells were treated with endothelin and/or PDTC. ChIP assays were performed with anti-MKL1. (I–L) RAW cells were transfected with siRNA targeting MKL1 or SCR followed by treatment with endothelin. ChIP assays were performed with anti-RelA (I), anti-acetyl H3 (J), anti-H3K4Me3 (K), and anti-BRG1 (L). Data represent mean ± SD. *p < 0.05, One-way ANOVA with post hoc Scheffe test.