Figure 2

p53 and NF-κB cooperate to induce mir-21. (a) Primary neonatal rat cardiac fibroblasts were treated with or without DFX and the NF-κB inactivator (NFI; 1 μM quinazoline) and mir-21 was quantified using the TaqMan miRNA assay. (b) Nuclear extracts from cardiac fibroblasts with or without DFX and NFI as in (a) were isolated and western blotted (WB) for p53 and RELA. Splicing factor 2 (SF2), was used to confirm equal protein loading. { (c) GIS-luciferase (GIS-Luc) and TK-renilla control (RL) were transfected into p53^-/- MEF cells with or without plasmids encoding p53 or RELA, and incubated with or without NFI as indicated. Firefly luciferase gene reporter activity was normalized to renilla control. (d) GIS, GIS with an AAA mutation engineered into the putative NF-κB binding site (GISmAAA), and GIS with the NF-κB binding site deleted (GISdel) were cloned upstream of firefly luciferase. Constructs were transiently transfected together with a TK-renilla luciferase plasmid and plasmids encoding p53 and RELA into p53^-/- MEF cells and firefly luciferase reporter gene activity (FL) was quantified and normalized against renilla (RL). Results represent a fold-difference between the three different GIS-constructs. (e) RelA^-/- (-/-) MEF cells and RelA^-/- MEF cells that were reconstituted with RELA using lentiviral overexpression (RA) were treated with or without DFX, and mir-21 quantification was performed. (f) RelA^-/- and reconstituted RelA^-/- MEF cells were treated with or without DFX, and whole cell (WCE) and nuclear (NE) extracts were western blotted for MDM2 (arrow), BAX and RELA. RhoGDI and SF2 were used to demonstrate protein loading. miRNA quantification is shown as mean ± standard error, at least n = 3. Luciferase reporter assays are presented as mean ± standard error for at least four independent replicates. Asterisks represent P < 0.05 (paired t-test).