FIGURE 2

Influence of microRNA let-7b on cyclins D1, D3, Cdk4, and cyclin A expression in melanoma cells. (A) SK-Mel-147 or G361 melanoma cells were transiently transfected with 30 nM of let-7b or control (co) precursor microRNA. Total protein extracts of cells were subjected to immunoblotting of cyclins D1, D3, Cdk4, and cyclin A. Immunoblots were re-probed with an anti-tubulin mouse monoclonal Ab to verify equal loading of proteins. (B-D) Immunofluorescence analyses of SK-Mel-147 melanoma cells transfected with 30 nM let-7b or control precursor microRNA. At 36 h after transfection melanoma cells were fixed with paraformaldehyde and stained with anti-cyclin D1 (B), anti-Cdk4 (C), or anti-cyclin D3 (D) antibodies. An Alexa Fluor 488 goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody was used as secondary antibody. Cells were counterstained with DAPI. Upper panel shows microscopic pictures of immunofluorescence staining of one representative experiment ( 100 and 400 magnifications, respectively). Lower panel shows graphic presentation of mean fluorescence intensities of three independent experiments. Asterisks in (B-D) indicate statistical significance of differences in cyclin D1, Cdk4, or cyclin D3 expression in let-7b-transfected cells compared with control precursor microRNA-transfected cells (P < 0.05; Mann-Whitney-U test).