Melanoma Cell Line Combination Assays

Human melanoma cell lines M14, A375 and WM115 were obtained as previously described [9 (link), 12 (link)] and cultured in RPMI supplemented with 10% FBS. For combination assays, BRAFi has been used starting from the highest dose of 5 μM and then diluted 1:2 for 10 times; SPV122 was used at fixed dose of 1.25 μM. All the other experiments have been performed with the following doses: 150 nM for BRAFi, 75 nM for MEKi and 1.25 μM for SPV122. Long-term colony formation assays have been performed treating M14 melanoma cells two times a week with 250 nM of a BRAFi and at every time point cells have been fixed using crystal violet staining as previously done [10 (link)]. For clonogenic assays, Zidovudine and Stavudine NRTIs and SPV122 and Efavirenz NNRTIs have been tested starting from 100 μM and then diluted 1:2 for 10 times. Encorafenib as BRAFi, MEK162 as MEKi, Efavirenz, Stavudine and Zidovudine have been obtained by Selleckchem. SPV122 was prepared as described [29 (link)].