cell motility and invasiveness. Moreover, our time lapse micros- copy data have clearly indicated that Sema 3A significantly reduced the migration of melanoma cells. Earlier it has been reported that p53 inhibits lung metastasis in B16F10 cells [50]. We have also observed that overexpression of Sema 3A augmented the activation of p53 in various melanoma models. Moreover, we have correlated the Sema 3A and p53 phosphorylation at Ser-15 in melanoma clinical specimens. Therefore, the inhibitory effect of Sema 3A on melanoma cells may be p53 dependent, although extensive study is required to understand such mechanism. Furthermore, our allograft data have shown that Sema 3A overexpression drastically reduced in vivo melanoma growth and metastasis. Moreover, attenuation of tumor growth by intratumoral injection of CM of clone 2 indicates that tumor secreted Sema 3A also suppressed tumor growth via paracrine mechanism. In recent time, treatment of cancer patients with anticancer agents/drugs (cancer chemotherapy) has shown greater promises; although there is some limitation of such therapy. Drug resistance of cancer cells has been known as the major burden for cancer chemotherapy and exhibit frequent clinical problem in patients. Therefore, development of novel therapeutic approach to overcome the drug resistance and increase the drug sensitivity of cancer cells remains a major challenge for the successful chemotherapy of cancer. In this study, we have noted that overexpression of Sema 3A in presence of various pharmacological anti-cancer agents (curcumin and DTIC) decreased cell survival as compared to control B16F10 cells. Moreover, we have observed that curcumin, even at comparatively lower doses significantly promotes apoptosis in Sema 3A overexpressed cells. Our live cell imaging data also suggested that fraction of control cells were escaped from apoptosis when they were incubated with curcumin (data not shown). Taken together, our experimental observations indicated that Sema 3A has no significant effect on melanoma cell survival but it increases the drug sensitivity of B16F10 cells. This study highlights that Sema 3A attenuates the metastatic signature and angiogenic switch in melanoma model which ultimately suppresses melanoma progression. The data revealed that Sema 3A increases drug sensitivity of melanoma cells. The results demonstrate that chemotherapy of cancer by anti-cancer agents along with combination of Sema 3A could be a rational and promising approach for the treatment of cancer. The study suggests that Sema 3A regulated pathway may act as potentially important therapeutic target for the management of malignant melanoma. Supporting Information Figure S1 Expression profile of Sema 3A in human melanoma cells and its role in melanoma migration and invasion. (A) Total RNA was isolated from human melanoma cells (A375 and SK-Mel-28) and Q-PCR analysis of Sema 3A expression was performed. Bar graph represents the normalized Sema 3A mRNA with GAPDH. *p,0.001. (B) Representative photographs of migrated/invaded melanoma cells were shown in S1B and the bar graphs were shown in Fig. 3B. (C) Invasion assays were performed with A375 and SK-Mel-28 cells either in absence or presence of Sema 3A recombinant protein. Invaded cell were counted and represented in the form of graph (Fig. S1C, panels I & II). (TIF) Figure S2 Sema 3A inhibits melanoma migration and melanoma-endothelial cell interaction through para- Semaphorin 3A Attenuates Melanoma Progression crine mechanism. (A) Representative photographs of migrated B16F10 cells showing Sema 3A abrogates melanoma migration through paracrine manner as described in Fig. 3C. (B) Photographs of migrated and invaded HUVEC showing Sema 3A attenuates melanoma-endothelial interaction as shown in Fig. 3D. (TIF) Figure S3 Immunohistochemical analyses of human normal skin biopsy and malignant melanoma tissues by using anti-phospho-p53 antibody. Two normal skin biopsy (A) and three malignant melanoma (B) specimens were analyzed immunohistochemically for visualizing the expression of Sema 3A (Fig. S3A, panels a, b & Fig. S3B panels a–c) and phospho-p53 of Ser-15 (Fig. S3A, panels e, f & Fig. S3B, panels g– i). Nuclei were stained with DAPI (Cy2 in green, Cy3 in red and DAPI in blue). (TIF) Figure S4 Sema 3A controls melanoma cell prolifera- tion. SK-Mel-28 cells were plated on coverslip in 24 well plates. The cells were treated with 100 ng/ml Sema 3A for 24 h followed by incubation with complete media supplemented with BrdU for another 24 h. Cells were stained with BrdU labeling and detection kit, visualized under fluorescence microscope, counted and photographed (A) at 106 magnification and represented in the form of bar graph (B). *p = 0.0022. Nuclei were stained with DAPI. (TIF) Figure S5 Time lapse videograph of wound migration of B16F10 cells. Confluent monolayer of B16F10 cells was wounded and the migration of the cell towards the wound was monitored and photographed under Nikon time laps microscope in an interval of 10 min up to 18 h and represented in the form of video using Image Pro-Plus software after compiling the images into a movie. (MP4) Figure S6 Time lapse videograph of wound migration of Sema 3A overexpressed B16F10 cells (clone 2). Confluent monolayer of clone 2 cells was wounded and the migration of the cell towards the wound was monitored and photographed under Nikon time laps microscope in an interval of 10 min up to 18 h and represented in the form of video using Image Pro-Plus software after compiling the images into a movie. (MP4) Figure S7 Time lapse videograph of wound migration of B16F10 cells treated with conditioned media (CM) of Sema 3A overexpressed B16F10 cells (clone 2). Confluent monolayer of control cells was wounded, treated with CM collected from clone 2 cells and the migration of cells toward the wound was monitored and photographed under Nikon time laps microscope in an interval of 10 min up to 18 h and represented in the form of video using Image Pro-Plus software after compiling the images into a movie. (MP4) Author Contributions Conceived and designed the experiments: GCK GC. Performed the experiments: GC SK RM. Analyzed the data: GC SK. Contributed reagents/materials/analysis tools: GC GCK. Wrote the paper: GC SK GCK. Analysis of histopathology and immunohistopathology data: TVP. PLoS ONE | www.plosone.org 12 March 2012 | Volume 7 | Issue 3 | e33633