TUNEL positive cells were counted and quantified as described

detection system with the KAPA SYBRH qPCR Kit according to the manufacturer’s instructions. The conditions for real-time PCR amplification were as follows: 2 min at 50uC, 1 min at 95uC, 40 cycles at 95uC for 3 s and 60uC for 1 min, followed by melting curve analysis at the end of each run from 60uC to 95uC. The primers used were as follows: Dph3, MMP-9, b-actin was significantly decreased compared with the cells transfected with scrambled siRNA. Transfected cells were then subjected to wound healing and transwell assays to evaluate their migratory potential. Both B16F10 and B16F0 cells transfected with a control siRNA were able to close a wound by 24 h. However, the wound inflicted on cells transfected with Dph3 siRNA had not yet closed up at this time. Consistently, results from transwell assay 4 Dph3 Potentates the Metastasis of Melanoma Cells also showed that the cell number of siDph3 cells moved across the membrane was much fewer than the siCTL cells. Taken together, these results indicated that silencing of Dph3 could reproduce the effect of Dph3 disruption by pDisrup 8 plasmid and drastically reduced the cell motility of melanoma cells. To determine ML-128 manufacturer whether Dph3 is widely expressed in melanoma cells, we examined the expression of Dph3 in various human melanoma cells. As expected, we found that Dph3 is highly expressed in examined human melanoma cells, especially in M102 cell line. Furthermore, in order to determine the cellular specific effect of Dph3 on tumor metastasis, we transfected human colon cancer cell HCT116, human ovary cancer cell A2780 and human skin cancer cell A431 with siDph3 plasmid and siCTL plasmid, Dph3 Potentates the Metastasis of Melanoma Cells followed by examination of the migration of these cell lines with transwell assay or wound healing. As shown in figure S1, siDph3 only affects the migration of A431 cells and has no effect on the migration of HCT116 and A2780 cells. These results suggested that Dph3 mainly affects the migration of some skin cancer cells. To further confirm the role of Dph3 in melanoma motility, we cloned Dph3 into pIRES-EGFP vector and transfected it into melanoma B16F10 and F0 cells. The transfection efficiency was confirmed by the expression of green fluorescence protein . Due to lack of effective antibody against Dph3, we further confirmed the overexpression of Dph3 by western blot with a Myc-tag antibody. The migration of Dph3 over- expressed cells was then examined by wound healing assay and migration assay. As shown in Dph3 Knockdown Reduces the Metastasis of Mouse Melanoma B16 F10 in vivo To further investigate the role of Dph3 in the metastasis of melanoma cells in vivo, an experimental metastasis assay was performed. Control and Dph3 knock down cells were injected into the lateral tail vein of C57BL/6J mice. 2 weeks post inoculation, animals were sacrificed and all the major organs were checked for the generation of tumor metastasis. The tumor metastasis was mainly observed in the lungs as previously reported. We found that injection of B16F10 control cells resulted in the formation of numerous lung colonies whereas silencing of Dph3 significantly suppressed pulmonary metastasis and only generated one third of lung colonies . In addition, B16F10 control cells produced nodules that occupied a higher percentage of the total lung area, while metastatic nodules of siDph3 cells generated discrete black foci. These results implied that Dph3 silencing indeed perturbed the metastasis