Fig. 3

The intermediate filament Nestin is a molecular target of miR-4443 and miR-4488 in melanoma. a Schematic representation of the entire 3’UTR of mRNA relative to nestin containing the two seed regions for miR-4488 and for miR-4443 located at positions 197–203 and 336–342, respectively (source miRWalk3.0). b, c To perform luciferase assays, HEK-293 cells were plated in a 6-wells plate and then co-transfected with a plasmid containing the 3’ UTR of nestin and Renilla luciferase plasmid together with miR-4443 (b), miR-4488 (c) or the relative scrambled (SCR). After 48 h of transfection, cells were harvested and luminescence was evaluated using Dual-Luciferase® Reporter Assay System. The values were calculated as fold change (± SD) compared to SCR considered as 1. d A375 SEN cells were transiently transfected with miR-4443, miR-4488 or with the SCR for 72 h. Cells have been then harvested and lysed to extract total proteins that have been subjected to Western blot analyses with the indicated antibodies (left panel). Tubulin levels have been estimated for the protein equal loading and densitometric analyses were performed using ImageJ software (right panel). Results were expressed as mean values from three independent experiments. The values were calculated as fold change (± SD) compared to SCR considered as 1. e A375 SEN cells transfected as described above have been stained with an antibody PE mouse anti-nestin for 45 min to perform flow cytometry analyses. Non-stained cells were used as negative control. Black arrows indicate the peaks relative to cells transfected with miR-4443 or miR-4488 mimics (left graphs). Quantitative analyses were calculated as “fold change” (± SD) compared to SCR considered as 1 (right graph). Data were analyzed using CytExpert version 2.2 software. f Representative images by confocal microscopy of nestin and DAPI immunostaining of A375 SEN cells transiently transfected as described above. Magnification 63x. All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value < 0.05 was considered as significant (Student’s t-test)