Immunofluorescence study Cells (control and clone 2) were grown on fibronectin coated cover slips for 6 h. After 6 h, cells were fixed with 2% paraformaldehyde and stained with FITC-conjugated phalloidin and visualized and photographed under fluorescence microscope (Zeiss). In separate experiments, B16F10, clone2 and SK-Mel-28 cells were plated on cover slips and incubated in serum free media for 24 h. SK-Mel-28 cells were either treated with Sema-3A recombi- nant protein (100 ng/ml) or vehicle for 24 h. Cells were fixed with 2% paraformaldehyde, stained with anti-Ser15-phospho-p53 anti- body and visualized under confocal microscope (Zeiss). Nuclei were stained with PI. To analyze the effect of curcumin on nuclear morphology, equal number of cells (control B16F10 and clone 2) were plated on cover slips and treated with indicated concentrations of curcumin. Cells were fixed and stained with propidium iodide as described earlier and visualized under fluorescence microscope [37]. Co-migration and co-invasion assays Invasion assay was performed in matrigel coated invasion chambers (BD Bioscience) using control B16F10 and clone 2 cells as described [38]. Briefly, B16F1, B16F10 or clone 2 were added to the upper portion of the Boyden chamber and incubated at 37uC for 18 h. In separate experiments, conditioned media collected either from clone 2 (Sema 3A overexpressed) or Sema 3A siRNA transfected B16F1 or B16F1 (with or without Sema 3A antibody) were used in the lower chamber as chemoattractant for B16F10 migration. In another experiments, A375 and SK-Mel-28 cells, treated with 100 ng/ml of Sema 3A were used in the upper portion of invasion chambers. FBS was used as chemoattractant. The cells invaded to the reverse side of the filter were stained, photographed, counted in 3 high-power fields (C/HPF) under an inverted microscope (Nikon), analyzed statistically and represented in the form of bar graph. The endothelial-melanoma cell interaction was shown by direct co-migration or co-invasion assays as described [28]. Briefly, HUVEC were added in upper chamber and control B16F10 or clones 2 cells were used in lower chamber. Anti-NRP1 blocking antibody was used in upper chamber. In separate experiments, conditioned media collected either from clone 2 (Sema 3A overexpressed) or Sema 3A siRNA transfected B16F1 or B16F1 (with or without Sema 3A Ab) were used in the lower chamber. FBS serve as chemoattractant for HUVEC migration and invasion. After 18 h, the migrated or invaded HUVEC in the reverse side of the filter were stained, photographed, analyzed statistically and represented graphically. BrdU incorporation assay SK-Mel-28 cells were seeded on coverslip and 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 fixed with 100% cold methanol and stained with BrdU labeling and detection kit (Roche), visualized under fluorescence microscope, photographed and analyzed. Wound assay and time laps microscopy To further confirm the motility of control B16F10 and clone 2 cells, wound assay was performed as described [28,36]. Briefly, clone 2 cells either alone or treated with anti-Sema 3A or anti- NRP1 blocking antibody were used for wound assay. In separate experiments, control B16F10 cells either alone or treated with conditioned media obtained from clone 2 were used. After 18 h, wound photographs were taken under inverted microscope (Nikon). The wound migration was also visualized under time laps Semaphorin 3A Attenuates Melanoma Progression microscopy. Confluent monolayer of cells was wounded and the migration of the cells 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. To further validate the effect of endogenous Sema 3A on melanoma migration, B16F1 cells were either silenced using Sema 3A specific siRNA or pretreated with Sema 3A blocking antibody and analyzed for wound assay. Migration assay was also performed using A375 and SK-Mel-28 cells treated with Sema 3A (100 ng/ml) for 18 h. MTT assay The effect of Sema 3A on melanoma cell proliferation was determined by MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyl tetrazolium bromide) assay. Briefly, control B16F10 or clone 2 cells were plated and allowed to grow at 37uC. Both cells were treated with Dacarbazine (0–400 mM) and curcumin ((0–50 mM) and MTT was added to the cells and incubated at 37uC for 4 h. The isopropanol was added to the cells, absorbance was measured in an ELISA reader (Bio-Rad) at 570 nM. DNA ladder assay Both control B16F10 and clone 2 cells either untreated or treated with two different concentrations of curcumin were incubated at 37uC for 12 h, lysed in ladder assay buffer (10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA, 0.1% SDS and 50 mg/ml Proteinase K) and genomic DNA was isolated by isopropanol precipitation method. Equal amount of DNA was analyzed by 2% agarose gel electrophoresis. In vivo tumorigenicity, in vivo invasion, histopathology and immunohistochemistry Control B16F10 and clone 2 cells (16106) were injected subcutaneously into the dorsal flank region of C57BL/6 male mice (6–8 weeks old). In separate experiments, serum free conditioned media collected from clone 2 cells was injected twice a week into the tumor site of mice generated by control cells. Tumor length and width were measured every week. After 4 weeks, mice were sacrificed, photographed, tumors were dissected out, weighted and used for histopathological and immunohisto- chemical studies. The mice were also dissected ventrally, photographed and the metastasized organs such as liver, intestine and kidney were used for histopathological studies. In vivo metastasis study by intra-cardiac injection Control B16F10 and clone 2 cells (16105) were injected intracardiacly into anesthetized C57BL/6 male mice (6–8 weeks old). After 3 weeks, mice were sacrificed; photographed, metasta- sized organs (lung, liver and intestine) were dissected out and used for histopathological studies. Statistical analysis The cell migration, invasion, MTT assays, tumor-endothelial interaction assays, tumor weights and volumes were analyzed statistically. Statistical differences were determined by paired Student’s t test. Differences were considered significant when P,0.05. Results Expression profile of Sema 3A in human melanoma clinical specimens and its correlation with melanoma growth Clinicopathological studies on human melanoma specimens have shown that melanoma progression is associated with angiogenesis PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33633