In vitro drug screen and validation

For the drug screen performed in RIG cell lines MAF-145 and MAF-496, the Approved Oncology Drugs Set VI (National Cancer Institute), comprising 129 drugs, supplemented by selinexor (Karyopharm Therapeutics) and AZD2014 (Astra Zeneca) was used. The complete list of drugs included in the screen is given in Supplementary Data ¹⁷. Cells were plated at a density of 5000 cells per well in 90 μL medium in a 96-well treated cell culture plate (Corning #3595) and allowed to adhere overnight. Drugs were applied in 10 μL of medium/1% DMSO at a concentration of 10 μM, resulting in a final concentration of 1 μM and 0.1% DMSO. Cells were incubated in the drug for 5 days. DMSO (0.1%) was used as a control. Cell viability was assayed after 5 days of treatment, using incubation with tritiated thymidine and quantification using a scintillation counter. Results were collected as counts/min and converted to survival by using the formula (sample – medium)/(DMSO – medium), where “sample” is the scintillation count for each drug-treated sample, “medium” is the scintillation count for a well containing medium only, and “DMSO” is the scintillation count for a three-well average of cells treated with 0.1% DMSO only (Supplementary Data ¹⁷). The drug screen was conducted twice in MAF-145 cells and once in MAF-496 cells due to limitations on cell availability.

Validation tests of single drugs were conducted using drug concentrations ranging from 0.316 nM to 10 μM in half-log₁₀ increments. Cells were plated as described above and incubated in a drug for 120 h. Three biological replicates were used for each drug concentration. Results were assessed using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer’s instructions. Survival was computed as above. IC₅₀ values were calculated using a variable-slope four-parameter non-linear model with maximum survival constrained at 100% (Prism 7, Graphpad).