4.2. Cell Culture and Viability Assay

Human cutaneous melanoma cell lines WM-3451 and WM-3623 were purchased from The Wistar Institute (Philadelphia, PA, USA), SK-Mel-2 cells were purchased from ATCC (Manassas, VA, USA) and SK-Mel-19 and SK-Mel-147 were obtained from Dr. Maria Soengas at the University of Michigan and have been described previously [20]. Cells were cultured in DMEM (Life Technologies, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1× antibiotic-antimycotic solution (Life Technologies). Cells were expanded and frozen immediately prior to authentication and then thawed only two to three months before experiments. Short tandem repeat profiles were performed on SK-Mel-19 and SK-Mel-147 cell lines (Genewiz, South Plainfield, NJ, USA). The profiles obtained do not match any established published profiles, and we were unable to identify published profiles for SK-Mel-19 or SK-Mel-147. NRAS exon 3 and BRAF exon 15 were PCR amplified from genomic DNA and subjected Sanger sequencing (MSU Genomics Core). The mutation status of the cells used were NRAS^Q61L (SK-Mel-2, SK-Mel-147, WM-3451), NRAS^Q61K (WM-3623), BRAF^V600E (SK-Mel-19). For viability assays, 1000 cells in 20 µL of DMEM containing 10% FBS were seeded into 348-well white bottom plates. Four to six hours later, 10 µL of 4× compound was added along with an additional 10 µL of either 4× second compound or media. After 72 h, 20 µL of CellTiterGlo (Promega) was added to each well. The assay plate was centrifuged at 300× g for 3 min. Luminescence was measured using a BioTek Synergy Neo plate reader. Data were normalized to values obtained for the vehicle-treated cells. Data were plotted as average values of at least three independent experiments. Non-linear least square analysis was used to fit data to a 4-parameter log-[inhibitor] vs. response curve using GraphPad Prism versions 6–8 (GraphPad Software, La Jolla, CA, USA).

The Loewe Excess synergy score and graphical outputs were determined using the online Chalice software (cwr.horizondiscovery.com, accessed on 17 March 2021) which implements the Chou–Talalay method [54] for Loewe Excess calculations from concentration–response data. This method is currently available in the downloadable (https://sourceforge.net/projects/combenefit/, accessed on 17 March 2021) freeware Combenefit software [55].