p53-NF-κB mediated mir-21 expression is dependent on STAT3. (a) Cardiac fibroblasts were treated with DFX with or without an inhibitor of STAT3 DNA binding (S3I-201), and cell lysates were incubated with streptavidin-coated beads on which biotinylated GIS duplex (oligo pulldown) or a scrambled sequence duplex (scrambled) was immobilized. Proteins bound to these duplexes were eluted and western blotted (WB) for STAT3, RELA and p53. (b) p53-RELA sequential ChIP was performed on cardiac fibroblasts with or without DFX and S3I-201. Results are presented as mean ± standard error for three independent experiments performed in triplicate. (c) Cardiac fibroblasts were treated with or without DFX and S3I-201, and mir-21 was quantified using the TaqMan miRNA assay. (d) Nuclear extracts (NE) from cardiac fibroblasts with or without DFX and S3I-201 were isolated and western blotted for STAT3, RELA and p53. (e) Using cell lysates from cardiac fibroblasts treated with DFX with or without S3I-201, RELA or control IgG immunoprecipitation (IP) was performed and western blotted for p53, STAT3 and RELA. (f) Stat3-/-MEF cells and Stat3-/-MEF cells that were re-constituted with wild-type Stat3 were treated with or without DFX and quantification for mir-21 was performed. All miRNA quantification results are presented as mean ± standard error, from three independent experiments and performed in triplicate. Asterisks represent P < 0.05.