Melanoma Cell Line Analysis

The human melanoma cell lines A375, MeWo, LOX IMVI, and WM793 were obtained from the American Type Culture Collection (ATCC), and WM 266.4 was provided by Dr. Michael Davies (MD Anderson). Cells were cultured in Dulbecco’s modified Eagle medium containing 5% fetal bovine serum at 37°C and in 5% CO2 and 95% air. For DETA NONOate treatment, cells were cultured to 75% confluency, and freshly prepared DETA NONOate stock was added to the final concentration as indicated in the text. After treatment times, cells were washed twice with cold phosphate-buffered saline and were harvested for analysis of pAKTS473 levels, AKT kinase activity, AKT substrate phosphorylation, and PTEN activity. An iNOS expressing construct was established as described by us previously (10 (link)). Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s specifications.