Fig. 3
TGF-β and STAT3 upregulation in miR-17-92 miRNA-deficient cells. a Immunoblot analysis on indicated proteins using protein lysates from cells cultured in DMEM containing 10% FBS. Levels of p-Smad2, Tgfbr2, and p-Stat3 (arrows) are increased in Mir17-92:Mir106b-25-deficient limb bud cells (17 dKO). b Quantification of the p-Smad2 to total Smad2 (t-Smad2) and p-Stat3 to total Stat3 (t-Stat3) protein ratios based on western blot data (n = 3, *p < 0.05). c Increased TGF-β signaling in 17 dKO cells upon stimulation. Serum-starved cells were treated with 50 ng/ml TGF-β1. Smad2 phosphorylation was analyzed at the indicated time points. d Relative mRNA expression of known targets of miR-17-92 miRNAs in 17 dKO limb bud cells (n = 3, *p < 0.05). e Luciferase reporter assay for miR-17 regulation on Tgfbr2 binding site. Primary limb bud cells were co-transfected with control miRNA mimic (Ctrl miR) or mmu-miR-17-5p (miR-17) and a luciferase reporter construct carrying a wildtype (Wt 3′UTR) or mutated 3′UTR (Mut 3′UTR) sequence of mouse Tgfbr2. The predicted binding sequence of Tgfbr2 3′UTR and relative luciferase units (RLU) are shown (n = 6, *p < 0.001)
