Cell viability assay

For IC50 measurement using the FGFR inhibitors, cells were dissociated into single cells and seeded into a 384-well tissue culture plate, each well with 200 viable cells and 40μL of growth medium. After 24 hours, compounds were added to each well over a 15-point concentration range, along with DMSO controls, using a Tecan D300e digital drug dispenser. Cells were cultured for 5 days in the presence of compound before assessing viability by adding 15μL of Cell Titer-Glo (Promega) to each well, incubating for 20 minutes at room temperature on a shaker, and measuring luminescence using an Envision plate reader. Each condition was performed in 5 replicates, and each dose point was normalized to DMSO controls to estimate relative viability. At least 2 independent experiments were performed for each compound and cell line. IC50 values were determined by GraphPad Prism using a 4-parameter dose-response model. Crystal violet staining assays were done by seeding cells into 6-well plates one day before addition of drug. Cells were grown in the presence of drug for four days (CCLP-1 cells) or two weeks (ICC13–7 cells), then washed with PBS, fixed with cold methanol, stained with 0.5% crystal violet solution (Sigma-Aldrich), and washed extensively under tap water.