Semaphorin 3A Attenuates Melanoma Progression compared to A375 cells, however, treatment of exogenous Sema- 3A significantly suppressed invasion in SK-Mel-28 as well as A375 cells (Fig. S1C). Taken together, these data showed that exogenous Sema 3A inhibits migration and invasiveness of human malignant melanoma cells. Moreover, we have observed drastic reduction of invasion of Sema 3A overexpressed melanoma cells (clone 2) as compared to control (Fig. 3A, panel I). The data were quantified and represented in the form of bar graph (Fig. 3A, panel II). Sema 3A abridged in vitro melanoma cell motility through autocrine and paracrine manner To determine the effect of Sema 3A on melanoma cell motility, wound assay was performed as described [28]. The data indicated that overexpression of Sema 3A significantly attenuated in vitro melanoma cell motility (Fig. 2B). However, treatment of clone 2 with anti-Sema 3A or anti-NRP1 blocking antibody drastically induced cell migration as compared to clone 2-derived cells alone demonstrating that tumor derived Sema 3A inhibits tumor cell motility through NRP1 dependent autocrine manner (Fig. 2B). Conditioned media collected from clone 2 significantly suppressed wound migration of control B16F10 cells (Fig. 2B) further suggesting that tumor derived Sema 3A could also suppress tumor cell motility via paracrine mechanism. On the other hand, silencing endogenous Sema 3A or blocking Sema 3A activity in B16F1 cells showed enhanced cell migration (Fig. 2C). These data further demonstrated the significance of loss-of-function of Sema 3A in melanoma cell migration. Figure 3. Sema 3A inhibits melanoma cell migration and invasion through autocrine and paracrine mechanisms. (A) Invasion assays were performed on matrigel coated invasion chamber. Cells (control or clone 2; 16105) were plated on upper chamber. After 18 h, invaded cells were stained with Giemsa and photographed (panel I), counted in 3 high-power fields (C/HPF) under an inverted microscope (Nikon), analyzed statistically and represented in the form of bar graph (panel II, *P = 0.022). (B) Autocrine mechanism of Sema 3A action on melanoma motility and invasiveness were demonstrated by migration and invasion assay using either B16F10 or clone 2 or B16F1 cells. *p,0.001, $p = 0.007, #p = 0.003 vs. B16F10. (C) Boyden chamber migration assay was performed using B16F10 cells to demonstrate the paracrine mechanism of action of Sema 3A. Conditioned media (CM) collected from clone 2 or B16F1 cells alone or B16F1 transfected with Sema 3A siRNA were used in the lower chamber as a chemoattractant. In separate experiments, either B16F10 cells treated with Sema 3A (100 ng/ml) or CM from B16F1 treated with Sema 3A antibody were used in the lower chamber. Migrated cells were counted, analyzed statistically and represented graphically. *p = 0.025, **p = 0.006 and $p = 0.011 vs. control, #p,0.001, ##p = 0.001. (D) Tumor-endothelial interaction was performed in modified Boyden chamber or matrigel coated invasion chamber. Endothelial cells were placed on upper chamber whereas similar conditioned media (as of Fig. 3C) were used in the lower chamber. Migrated or invaded cells were stained with Giemsa, photographed, analyzed statistically and represented in the form of graph. *p,0.001 vs. control, **p = 0.004, #p = 0.01, $p = 0.013. doi:10.1371/journal.pone.0033633.g003 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33633