Figure 3: MTSS1 promotes melanocytic cell motility.

(a) In vitro scratch assay with human A375 melanoma cells 48 h after interference with MTSS1 expression by the indicated set of siRNAs. Images were acquired at 0, 24 and 48 h time points after scratching. Scale bar, 350 μm. (b) Representative experiment to rescue the invasive potential of human A375 melanoma cells into Matrigel by expression of wild-type (wt) and mutated (I-BARmut, WH2mut) MTSS1 isoforms after RNA interference with MTSS1 expression for 48 h. Relative invasion into Matrigel for 20 h as compared with expression of empty-vector (pEGFP-N1) alone by the two-tailed Student’s t-test, **P≤0.01, NS: not significant. Bars correspond to mean±s.d. of triplicate data points. (c) Left, immunoblotting for MTSS1 in HMEL cells stably expressing ectopic MTSS1 or control empty-vector. Right, migration of empty-vector and ectopic MTSS1-expressing HMEL cells through fibronectin-coated culture inserts for 20 h in the presence of PDGF. The assay was performed in triplicate. Bars represent the mean of five random microscopic fields±s.d. Data were compared using the two-tailed Student’s t-test, **P≤0.01. (d) Cell-spreading assay with human A375 melanoma cells for 45 min on fibronectin after 48 h RNA interference with MTSS1 expression as determined by the percentage spread of total cells. Bars represent mean±s.d. from three independent experiments. Data were compared with control-treated cells (Ct.siRNA) using the two-tailed Student’s t-test, *P≤0.05, **P≤0.01.