The antitumoral effect of miR-150-5p is diminished in E2F3^mut cells. (A) Protein expression of E2F3, c-Myc, and in PBL-1 (WT; gray) and MYC^KO (orange) cells showing the western blot plots (left) and relative expression (RQ; right) normalized to GAPDH. (B) Relative expression (RQ) of miR-150-5p in WT (gray), E2F3^mut (green), or MYC^KO (orange) cells. (C) Comparison of miR-150-5p expression when cells are transfected with NC miRNA (gray) or miR-150-5p mimic (blue) into WT, E2F3^mut, or MYC^KO cells, measured by RT-qPCR. (D) Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in NC miRNA and miR-150-5p mimic–transfected E2F3^mut cells. (E) Cell cycle stages were assessed by flow cytometry with TO-PRO-3 staining and flow cytometry at 72 hours in E2F3^mut cells transfected with NC miRNA or miR-150-5p mimic; figures show the percentage of cells in each stage. (F) Flow cytometry plots of apoptosis (left) evaluated by annexin V (Pacific Blue)/TO-PRO-3 (APC) staining 24 hours after transfection. Plots of relative viability of miR-150-5p transfected cells normalized to NC miRNA transfected ones; and percentage of apoptotic (early plus late) E2F3^mut cells. (G) Gene expression (RT-qPCR) of miR-150-5p targets in WT vs E2F3^mut transfected cells with NC or miR-150-p mimic. (H) Western blot plots of the protein expression of miR-150-5p targets in WT vs E2F3^mut transfected cells with NC or miR-150-p mimic. (I) Relative protein expression of miR-150-5p targets. All experiments were performed in biological triplicates and technical duplicates, unless otherwise specified. Statistical significance was determined by t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant.