Semaphorin 3A Attenuates Melanoma Progression [39]. In this study, we have analyzed 8 human melanoma tissue samples and 5 normal skin biopsy samples by histopathology and immunohistochemistry. The clinical samples were collected from local hospital with informed consent. The occurrences of melanoma in these samples were examined by H&E staining and the micrographs were taken in 106 magnification (Fig. 1A, panel a–e and 1B panel a–h). The expression level of Sema 3A in these samples was determined by immunohistochemistry using anti-Sema 3A antibody. The results revealed that significant expression of Sema 3A was observed in normal skin biopsy specimens (Fig. 1A, panel f–j). However, the level of Sema 3A was significantly reduced in all 8 melanoma tissue sections indicating that loss of Sema 3A level may be linked with melanoma growth (Fig. 1B, panel i–p). Taken together, these data suggested that loss of Sema 3A expression is associated with melanoma progression in human clinical specimens. Generation of Sema 3A clone in murine melanoma cells To examine the endogenous level of Sema 3A, we have used two different murine melanoma cell lines. Our RT-PCR and Western blot analysis data revealed that low metastatic melanoma cells (B16F1) express significantly higher level of Sema 3A as compared to highly metastatic (B16F10) cells (Fig. 1C and data not shown). To further study the level of Sema 3A in human melanoma cells (A375 and SK-Mel-28), Q-PCR was performed. The data showed that A375 cells have significantly higher Sema 3A expression than SK-Mel-28 cells (Fig. S1A). To investigate the role of Sema 3A on melanoma progression, we generated Sema 3A positive stable clone in highly metastatic B16F10 cells as described under material and methods. Our results revealed that among three hygromycin resistance Sema 3A clones, clone 2 expressed high level of Sema 3A as compared to clones 1 and 3 (Fig. 1D). Hence clone 2 is used for all further studies. Figure 1. Histopathological and immunohistochemical analyses of human normal skin biopsy and malignant melanoma tissues. (A&B) Five normal skin biopsy and eight malignant melanoma specimens were stained with hematoxylin and eosine and the histopathologic micrographs were visualized at 106 magnifications (Fig. 1A, panels a–e, & Fig. 1B, panels a–h). Tissue sections were also analyzed immunohistochemially for visualizing the expression of Sema 3A (Fig. 1A, panels f–j & Fig. 1B, panels i–p). Sema 3A was stained with Cy2 (Green). Note that significant loss of Sema 3A expression was detected in malignant melanoma tissue samples as compared to normal skin samples. (C) Endogenous expression of Sema 3A mRNA in B16F1 and B16F10 cells as detected by RT-PCR. GAPDH was used as loading control. (D) Expression of Sema 3A in B16F10 clones were analyzed by Western blot using specific antibody. The second clone (lane 3, denoted as clone 2) shows significantly higher expression of Sema 3A as compared to other clones and control B16F10 cells. The data represent three independent experiments exhibiting similar results. doi:10.1371/journal.pone.0033633.g001 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33633