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

Figure 4.

Figure 4

miR‐205 directly regulates LAMC1. (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 (upper panel). LAMC1, and Vinculin as loading control, were quantified by western blot (middle panel). Densitometric analysis of LAMC1 bands is reported in the lower panel. (B) Schematic representation of the interaction between miR‐205 and the binding site on the wild‐type LAMC1‐3′UTR and the mutated control. Luciferase activities (lower panel) for LAMC1‐3′UTR‐wt and LAMC1‐3′UTR‐mut plasmids were determined 24 h after transfection of HEK‐293 cells. The results are representative of 4 independent experiments. Bars indicate S.D. calculated on 3 different replicates (p < 0.05). (C) Phenocopy experiment showing the inhibition of colony formation obtained by transfecting MDA‐MB‐231 cells with a vector encoding a shRNA silencing LAMC1 (sh‐LAMC1) compared with the corresponding empty vector (sh‐CTR) and (D) Down‐modulation of LAMC1 protein (evaluated by western blot) 48 h after transfection.