Fig. 2

miR-4443 and miR-4488 transient overexpression affects migration/invasive potential and cytoskeletal remodeling of melanoma cells. a Total RNAs were extracted from A375 sensitive cells (SEN) and their resistant counterparts (RES) to perform qRT-PCR analyses for miR-4443 and miR-4488 expression levels. Each miRNA transcript was quantified using the comparative Ct method for relative quantification (ΔΔCt) using U6 as normalizer. The values were calculated as “fold change” (± SD) compared to SEN considered as 1. b A375 SEN cells were transiently transfected with miR-4443, miR-4488 or the relative scrambled (SCR) for 48 h. Transfected cells have been then harvested and cultured in the presence of double well culture inserts to perform wound healing assays. After 24 h, inserts were removed and each well was photographed at 10 × magnification (scale bar 500 μm) immediately after insert removal (T-0) and after 24 h (T-24H) (left panel). The open residual area (right panel) was measured by using the ImageJ software in oncomiR-transfected cells as compared to SCR-transfected ones. The values were calculated as “fold change” (± SD). c To perform migration assays, A375 SEN cells, transfected as described above, were seeded in serum-free media into the upper chamber of a transwell, whereas the lower chamber was filled with 10% FBS RPMI. After 8 h, cells remaining on the top side of the membrane were counted and the average number ± SD of cells are reported as fold change respect to control (SCR) considered as 100. d To perform invasion assays, A375 SEN cells, transfected as described above, were seeded into the upper chamber of a transwell coated with Matrigel, whereas the lower chamber was filled with 10% FBS RPMI. After 24 h, invading cells were counted and the average number ± SD of cells are reported as fold change respect to control (SCR) considered as 100. e, f Migration and invasion assays were performed as described above using A375 RES cells transiently transfected with the anti-miR-4443, the anti-miR-4448 or the SCR for 48 h before performing the appropriate assays. For all migration and invasion assays, three independent experiments were performed; each experiment was performed at least in quadruplicate. g Representative images by confocal microscopy of F-Actin and DAPI immunostaining of A375 SEN cells transiently transfected with miR-4443, miR-4488 or the SCR for 48 h. Red arrows indicate filopodia and stress fibers. Magnification 63x. h Quantification analyses of the long cell axis measured from the aforementioned confocal images were performed by Zeiss Zen control software. 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)