To further study the level of Sema 3A in human melanoma cells, Q-PCR was performed

cubated in serum free media for 24 h. SK-Mel-28 cells were either treated with Sema-3A recombinant protein or vehicle for 24 h. Cells were fixed with 2% paraformaldehyde, stained with anti-Ser15-phospho-p53 antibody and visualized under confocal microscope. Nuclei were stained with PI. To analyze the effect of curcumin on nuclear morphology, equal number of cells 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. 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 for 18 h. MTT assay The effect of Sema 3A on melanoma cell proliferation was determined by MTT assay. Briefly, SB 203580 supplier Control B16F10 or clone 2 cells were plated and allowed to grow at 37uC. Both cells were treated with Dacarbazine and curcumin 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 at 570 nM. Co-migration and co-invasion assays Invasion assay was performed in matrigel coated invasion chambers using control B16F10 and clone 2 cells as described. 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 or Sema 3A siRNA transfected B16F1 or B16F1 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 under an inverted microscope, 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. 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 or Sema 3A siRNA transfected B16F1 or B16F1 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. 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, 15523001” lysed in ladder assay buffer 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 were inj