Vemurafenib plx4032

All melanoma cell lines were obtained from the Massachusetts General Hospital Cancer Center. C32, K2, MMACSF, SKMEL28, and WM115 cell lines were grown in DMEM/F12 (Invitrogen) supplemented with 5% fetal bovine serum (FBS) and 1% sodium pyruvate (Invitrogen). COLO858, LOXIMVI, MZ7MEL, RVH421, and WM1552C cell lines were grown in RMPI 1640 (VWR) supplemented with 5% fetal bovine serum (FBS) and 1% sodium pyruvate (Invitrogen). We added penicillin (50 U/ml) and streptomycin (50 μg/ml) to all growth media.
Chemical inhibitors from the following sources were dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solutions for in vitro studies: vemurafenib (PLX4032), PLX4720, SB590885, selumetinib (AZD6244) and AZ628 (all from MedChem Express), JNK-IN-8 (EMD Millipore), SP600125, doramapimod (BIRB796), and SB202190, GDC0941, tofacitinib (CP-690550), and IKK16 (all from Selleck Chemicals).

A2058 cells were inoculated in a culture flask, and a high dose of vemurafenib (PLX4032, Cat#HY-L032) (MedChemExpress, USA) was used for treatment. When most of the cells had died, the dead cells were washed away and the medium was changed to a drug-free medium. Post reaching confluence, the cells were digested and re-inoculated into a new culture flask, and some cells were cryopreserved for RNA-seq. The cells were treated with high density vemurafenib until the cells reached 30% confluence. This process was repeated 3 times, and we obtained R0, R1, R2, and R3 cells, which were then used for RNA-seq analysis. Melanoma cells in the exponential growth phase were inoculated into culture bottles. After 48 h, the cells were transferred to a drug-free medium and expanded until the next mitotic phase. Then, the above steps were repeated using vemurafenib that was two times more concentrated. At the same time, cell death was observed every day, the fresh complete medium containing vemurafenib was replaced, and Cell Counting Kit 8 (CCK8) analysis was performed regularly until melanoma cells grew stably in the medium. This process lasted for six months, and transcriptome sequencing was performed once every two months; thus, 4 samples (R0, R1, R2, and R3) were obtained.

Human rhabdomyosarcoma (RD, ATCC, CCL-136™) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 100 units/mL of penicillin and 1000 µg/mL of streptomycin (Gibco, Waltham, MA, USA) as previously described [40 (link)]. Human EV-A71 viruses (isolate MY104-9-SAR-97, GenBank: DQ341368) were cultured in RD cells in serum-free DMEM supplemented with 2% FBS. Virus stocks were stored at −80 °C and were titrated by a 50% tissue culture infection dose (TCID50) assay as previously described [41 (link)]. Vemurafenib (PLX4032) and remedesivir (GS-5734) were purchased from MedChemExpress (MCE) and dissolved in dimethylsulfoxide (DMSO) to prepare 20 mM stock.