miRNA inhibition of HRG-dependent signaling and cell viability. MCF7 cells were transfected with the indicated miRNAs. (A) Three days after transfection, cells were either left untreated (0 min) or stimulated with 10 ng/ml HRG for the indicated times prior to lysis. Equal amounts of cell lysate were analyzed by immunoblotting using antibodies specific for ErbB3(pY1289), ErbB2(pY1221/1222), Akt(pT308), Akt(pS473), and Erk1/2(pT202/pY204). Membranes were further probed with antibodies that detect the total level of these proteins. Tubulin was detected to confirm equal loading. (B-D) Western Blot signals from (A) as a representative experiment were quantified by ImageJ. pAkt signals were normalized to total Akt (C), whereas phosphorylated Erk signals were normalized to tubulin (D) because total Erk levels were strongly affected by miRNA expression (see B; signals at 0 min HRG). The unstimulated control was set to 1. (E) One day after transfection, cells were plated into 96-well plates and grown in medium containing 0.5% FCS (− HRG) and 0.5% FCS supplemented with 5 ng/ml HRG (+ HRG), respectively. Five days later, cells were fixed and stained with crystal violet and the absorbance at 550 nm was measured. Data were normalized to the basal growth in 0.5% FCS. The mean ± SEM of 2–4 independent experiments is shown.