Fig. 1

A375 resistant melanoma cells have increased migratory/invasive phenotypes and altered cytoskeletal remodeling as compared to sensitive counterparts. a A375 sensitive cells (SEN) and their resistant counterparts (RES) were 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 RES cells as compared to SEN ones. The values were calculated as “fold change” (± SD). b To perform migration assays, A375 SEN and RES cells 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 considered as 100. c To perform invasion assays, A375 SEN and RES cells 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 considered as 100. Three independent experiments were performed (a, b, c); each experiment was performed at least in quadruplicate. d, e xCELLigence RTCA technology was used to measure cell migration and invasion of A375 SEN and RES cells. To evaluate migration (d), cells were seeded in serum-free medium on filters in the upper chamber, whereas the lower chamber was filled with 10% FBS RPMI. Cell migration was monitored for 6 h. To evaluate invasion (e), cells were seeded in serum-free medium on filters coated with Matrigel in the upper chamber, whereas the lower chamber was filled with 10% FBS RPMI. Cell invasion was monitored for 25 h. Each experiment was performed at least twice in quadruplicate (d, e). The slope represents the change rate of cell index values generated in a 0–1 h time frame. f, g Representative images by confocal microscopy of F-Actin and DAPI immunostaining of A375 SEN and RES cells that were cultured into 8-well μ-slides 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)