Semaphorin 3A Attenuates Melanoma Progression Overexpression of Sema 3A suppresses metastatic phenotype of B16F10 cells Earlier studies have shown that invasive behavior of melanoma cells is one of the key phenomena during melanoma progression [40]. Colony formation on matrigel has been frequently employed as a reliable assay to determine the in vitro tumorigenicity and metastatic phenotype of cancer cells [41,42]. To study the effect of Sema 3A on in vitro tumorigenicity of melanoma cells, matrigel colony formation assay was performed. The results revealed that clone 2 cells exhibits significantly reduced colony formation on matrigel as compared to control B16F10 cells (Fig. 2A, panel a). To investigate the role of Sema 3A on stress fibre formation, both the control B16F10 and clone 2 cells were grown in fibronectin coated plates and stained with FITC-conjugated phalloidin. The results showed that there was significant increase in actin stress fiber (indicated by arrow) and lamellipodia formation in control cells as compared to clone 2 (Fig. 2A, panel b). These data suggested that overexpression of Sema 3A attenuates in vitro metastatic phenotype of melanoma cells. Effect of exogenous Sema 3A on melanoma cell migration and invasion To determine the effect of exogenous Sema 3A on human melanoma cell migration and invasion, we have used two different human malignant melanoma cell lines, A375 and SK-Mel-28. Both these cells exhibited reduced migration when treated with recombinant Sema 3A (Fig. 2D). We have also observed that untreated SK-Mel-28 cells exhibit significantly higher invasion as Figure 2. Loss-and-gain of Sema 3A functions and it’s correlation with metastatic phenotype of melanoma cells. (A) panel a, equal number of control B16F10 and clone 2 cells (16104) were grown on matrigel coated plate and incubated at 37uC for 7 days. Colony photographs were taken at 106 magnification. Panel b, control B16F10 and clone 2 cells (16104) were plated on fibronectin coated coverslips and incubated for 6 h. Cells were fixed in 2% PFA and stained with FITC-conjugated phalloidin (green). Stress fibers were shown by arrows. Nuclei were stained with PI (red). Photographs were taken at 606 magnification. (B) The effect of Sema 3A on B16F10 cell motility was performed by wound migration assay. Control B16F10 or clone 2 positive cells were used for wound migration. In separate experiments, clone 2 positive cells either treated with anti-Sema 3A or anti-NPR1 blocking antibody were used for wound migration assay. In another experiments, control B16F10 cells were incubated with conditioned medium (CM) collected from clone 2 and wound migration assay was performed. After 18 h, cells were visualized and photographed under Nikon microscope at 106 magnification. (C) To further study the loss-of-function of Sema 3A in regulating melanoma cell migration, wound assay was performed using B16F1 cells. Cells were either transfected with Sema 3A siRNA or treated with Sema 3A blocking antibody and used for migration assay. (D) To study the gain-of-function, wound migration assays were performed using A375 or SK-Mel-28 cells treated with recombinant exogenous Sema 3A for 18 h. Sema 3A significantly attenuates wound motility of melanoma cells. All the data shown here are the representatives of triplicate independent experiments. doi:10.1371/journal.pone.0033633.g002 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e33633