Genome-wide miRNA screen. (A) Workflow of the screening procedure. MCF7 cells were transiently transfected in a 96-well format with a human mimic miRNA library comprising 879 human miRNAs. Three days post transfection, cells were either left untreated (basal) or stimulated with heregulin (HRG) for one hour followed by the detection of total Akt and phosphorylated Akt(pT308) levels by In-Cell Western. (B) MCF7 cells were transfected with the indicated miRNAs and siRNAs and subjected to In-Cell Western as described in (A). Akt activation after HRG stimulation (ΔpAkt) was calculated as described in the Methods section. One representative experiment performed with triplicate samples is shown. The data was analyzed by one way Anova followed by Tukey’s multiple comparison test (**p < 0.01, ***p < 0.001). (C) Workflow of the bioinformatic analysis of the screen data. For each replicate, the ratio of pAkt/Akt signal intensity and the difference between untreated and stimulated samples were calculated. miRNAs that significantly (p < 0.05) altered Akt activity and had values of ΔpAkt < 1 or ΔpAkt > 2 for both replicates were considered as screen hits. (D) Correlation of the ratios of phosphorylated Akt and total Akt signal intensities (pAkt/Akt) for the basal (left) and HRG-stimulated (right) condition. (E) Screen data visualization: Akt activation upon HRG stimulation (ΔpAkt) for each miRNA is plotted. The mean of the two replicates for each miRNA is presented in ascending order.