miR-149 suppresses heregulin signaling. (A) miR-149 recognition site in the ErbB3 3′UTR (upper panel). HEK293T cells were co-transfected with miR-con or miR-149 along with a luciferase reporter containing the wild-type (WT) or mutated ErbB3-UTR lacking the miR-149 seed region (mt; Δ527-533). The next day, cells were lysed and luciferase activity measured and normalized to the activity of the co-expressed Renilla reporter (lower panel). Data correspond to the mean ± SEM of four independent experiments performed with triplicate samples. Data were analyzed by one way Anova followed by Tukey’s multiple comparison test (**p < 0.01). (B) MCF7 cells were transfected with an ErbB3-specific siRNA pool (siErbB3), miR-con or miR-149, respectively. Three days post transfection, RNA was extracted and ErbB3 levels were determined by qRT-PCR. Values were normalized to GAPDH. Data are shown as the mean ± SEM of three independent experiments and analyzed by one-way Anova followed by Tukey’s multiple comparison test (**p < 0.01). (C, D) MCF7 cells were transfected with miRNAs and siRNAs as indicated and analyzed three days later. (C) Cells were lysed and ErbB3 expression analyzed by immunoblotting. Tubulin was detected as a loading control. (D) Cells were left unstimulated (0 min) or stimulated with 10 ng/ml HRG for the indicated times prior to lysis. Cell lysates were analyzed by immunoblotting using the indicated antibodies (E-G) Western Blot signals from two independent experiments were quantified by ImageJ. pAkt signals were normalized to total Akt (E), whereas phosphorylated Erk signals were normalized to tubulin because total Erk levels were strongly affected by miRNA expression (see G; signals at 0 min HRG). The unstimulated control was set to 1. The mean intensities ± SEM are shown.