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. 2012 Apr 19;6(4):458–472. doi: 10.1016/j.molonc.2012.03.003

Figure 3.

Figure 3

miR‐205 directly targets E2F1. (A) Transfection of MDA‐MB‐231 cells with 100 nmol/L pre–miR‐205 or a scrambled oligonucleotide and collection of RNA and proteins after 72 h and 96 h. miR‐205 expression was evaluated by real‐time PCR. E2F1 mRNA levels was evaluated by qRT‐PCR and protein levels were quantified by western blot. (B) Schematic representation of the interaction between miR‐205 and the binding site (conserved‐A and poorly conserved‐B) on the wild‐type E2F1‐3′UTR and the mutated control. (C) Luciferase activity for E2F1‐3′UTR‐wt, E2F1‐3′UTR‐mut‐A, E2F1‐3′UTR‐mut‐B and E2F1‐3′UTR double mutant (DM) plasmids was determined 24 h after transfection of HEK‐293 cells. The results are representative of four independent experiments. Bars indicate S.D. calculated on 3 different replicates (p < 0.01). (D) Phenocopy experiment showing the inhibition of colony formation obtained by transfecting MDA‐MB‐231 cells with a vector encoding a shRNA silencing E2F1 (sh‐E2F1) compared with the corresponding empty vector (sh‐CTR) and (E) Down‐modulation of E2F1 protein (evaluated by western blot) 96 h after transfection.