EC50 determinations and proliferation assays

Melanoma cell lines were tested by seeding 96-well plates at 3,000 cells per well. The next day, Calpain inhibitor I was added at concentrations from 1.25 μM to 160 μM in three replicates, with DMSO as a negative control. After 72 hours, cell proliferation was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). EC₅₀ values were determined using GraphPad Prism. The effect of Calpain inhibition combined by MEK inhibition on cell proliferation was tested by adding increasing concentrations of MEK inhibitor Trametinib (GSK1120210) (SelleckChem) from 1 pg to 10 μM and a constant concentration of Calpain inhibitor I (6 μM for A375, 76T, 108T) and 4 μM for 74T. Cells were evaluated for viability after 72 hours as described above. Statistical analysis was performed using Microsoft Excel to generate P values to determine significance (Student t test).