To further validate the suppressive effect of Sema 3A on real time melanoma cell motility, wound migration assay was performed using Time lapse microscopy under the same experimental conditions as described above. The video demon- strated that control B16F10 (18 h; Fig. S5) cells exhibit faster movement and complete closer of wound as compared to clone 2 cells (18 h; Fig. S6). Moreover, incubation of control B16F10 cells with conditioned media of clone 2 showed significant reduction of migration as well as exhibit similar motility phenotype like clone 2 cells (18 h; Fig. S7). These data further corroborate that clone 2 derived Sema 3A attenuates motility of control B16F10 cells. Taken together, the time lapse experimental data demonstrated that Sema 3A through an autocrine and/or paracrine manner inhibits melanoma cell motility and may act as potential suppressor of melanoma progression. To further demonstrate the role of Sema 3A in melanoma cell migration through autocrine and paracrine mechanism, Boyden chamber migration assay was performed where conditioned media (CM) collected from clone 2 or B16F1 cells (with or without Sema 3A siRNA transfection) were used in the lower chamber as chemoattractant. Moreover, B16F10 cells either treated with Sema 3A (100 ng/ml) or CM collected from B16F1 treated with Sema 3A antibody were also used in the migration assay. The data revealed that supplying exogenous Sema 3A can impede and silencing or inhibiting its activity can enhance B16F10 migration (Fig. 3C and Fig. S2A). These results demonstrated that Sema 3A regulates melanoma cell migration through autocrine (Fig. 3B and Fig. S1B) and paracrine (Fig. 3C and Fig. S2A) mechanism. Sema 3A attenuates melanoma cell proliferation To determine whether overexpression of Sema 3A exerts any effect on melanoma cell proliferation, MTT assay was performed. Equal number of control B16F10 and clone 2 cells were grown in serum free media for 24 h and then incubated with 0.5 mg/ml of MTT. The proliferation rate of control and clone 2 cells were analyzed by ELISA reader and plotted graphically (Fig. 4B). The data showed that overexpression of Sema 3A reduces the cell viability to 43% of the control. To further confirm this study, BrdU incorporation assay was performed using Sema 3A treated SK-Mel-28 cells. Cells were stained with BrdU labeling and detection kit, visualized under fluorescence microscope, photo- graphed, analyzed and represented in the form of bar graph. The data showed significant reduction in BrdU incorporation in Sema 3A treated cells (Fig. S4 A and B). Enhanced expression of Sema 3A augments p53 phosphorylation p53, a tumor suppressor protein plays crucial role in regression of cancer progression [43]. Recent studies have revealed that phosphorylation of Ser-15 residues of p53 exhibit growth retardation in melanoma [44]. Tedeschi et al reported that growth cone retraction by Sema 3A is overcomed by cGMP in wild type but not in p53 dorsal root ganglia [45]. In this study, we have observed that overexpression of Sema 3A inhibits in vitro tumorigenic phenotype of melanoma cells. Therefore, we sought to determine whether Sema 3A has any role in suppression of melanoma progression and the involvement of activated p53 in this process. Accordingly, control and clone 2 cells were analyzed by immunofluorescence using anti-phospho p53 (Ser-15) antibody and analyzed by confocal microscopy (Zeiss). The results indicated that cells overexpressing Sema 3A enhances p53 phosphorylation at Ser-15 residue (Fig. 4C, panel I) suggesting the possible involvement of activated p53 in Sema 3A regulated melanoma progression. To further validate our findings, SK-Mel-28 cells Semaphorin 3A Attenuates Melanoma Progression were treated with Sema protein (100 ng/ml) for 60 min and analyzed by immunofluorescence using anti-phospho p53 (Ser-15) antibody (Fig. 4C, panel II). The results indicated that exogenous supply of Sema 3A enhances p53 phosphorylation at Ser-15 residue. To further correlate p53 and Sema 3A in clinical samples, we analyzed the expression profile of Sema 3A and phospho-p53 in normal as well as malignant melanoma clinical specimens. These data suggested the enhanced expression of Sema 3A and phospho-p53 in normal skin samples as compared to malignant melanoma specimens (Fig. S3A and B). These findings further strengthened the correlation between p53 and Sema 3A in melanoma progression. Tumor-derived Sema 3A attenuates melanoma- endothelial cell interaction through NRP1 dependent paracrine manner Our earlier studies have demonstrated that tumor-endothelial interaction plays crucial role in tumor angiogenesis which ultimately promotes tumor progression and angiogenesis [28]. To study the role of Sema 3A in tumor-endothelial interaction through autocrine and paracrine mechanisms, co-migration and co-invasion assays were performed using HUVEC and melanoma cells. Conditioned media (CM) collected from clone 2 or B16F1 cells (with or without Sema 3A siRNA transfection) or treated with Sema 3A antibody were used in the lower chamber. Moreover, HUVECs were also treated with Sema 3A (100 ng/ml) and used in upper chamber for co-migration and co-invasion assays. The data depicted that providing exogenous Sema 3A can reduce and silencing or blocking endogenous Sema 3A can enhance HUVEC migration/invasion (Fig. 3D and Fig. S2B), suggesting that Sema 3A regulates melanoma-endothelial interactions through paracrine mechanism. Moreover, we have observed the involvement of NRP1 in tumor-endothelial interaction [28]. NRP1 is identified as one of the cell surface receptor that interacts with Sema 3A [30,31]. Therefore, to investigate the effect of Sema 3A on tumor- endothelial interaction, co-migration and co-invasion assays were performed as described in materials and methods. Our results revealed that cells overexpressing Sema 3A (clone 2) exhibit reduced migration and invasion of HUVEC towards tumor cells (Fig. 4A, panels I and II). However, blocking the endothelial cell- derived NRP1 has reversed these effects (Fig. 4A, panels I and II). Taken together our results suggested that overexpression of Sema 3A regulates tumor-endothelial cell interaction through NRP1 dependent paracrine mechanism. Sema 3A sensitizes melanoma cells in response to various anti-cancer agents To examine the effect of various anti-cancer agents in absence or presence of Sema 3A on melanoma cell death, cell viability assay was performed. Briefly, both control B16F10 and clone 2 cells were exposed with various anti-cancer agents like curcumin (0–50 mM) and Dacarbazine (0–400 mM) for 12 h and 24 h respectively, and the cell viability was determined by MTT assay. The results have shown that Sema 3A significantly sensitizes melanoma cells in response to these agents in a dose dependent manner (Fig. 5A & B). Curcumin selectively promotes apoptosis in response of Sema 3A Earlier we and others have reported that curcumin with higher doses significantly reduced cell viability and induce apoptotic phenotype in B16F10 cells [38,46]. In this study, we have observed that curcumin significantly suppresses the survival of Sema 3A PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33633