IC50 Analysis and MTT viability assay

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, (MTT) assay was used to determine the effects of pitavastatin on the viability of the non-responsive and responsive GSC populations. Briefly, 20,000 cells/well were plated in laminin-treated 96-well plates with 100uL of culture media. Following an initial 24hr incubation, the cells were treated with 100μL of culture media with pitavastatin at varying concentrations (0.0, 0.1, 0.6, 1.0, 3.0, 6.0, 10.0, 33.0μM) and incubated at 37C for four days. Vehicle amounts were adjusted such that the vehicle concentration in all conditions was equivalent to the maximum drug dosage tested (DMSO 0.2% v/v). Following the 4-day treatment, spent media was replaced with 100μL of serum-free culture media with MTT (0.5mg/mL) and incubated at 37°C for 60 minutes. Following incubation, supernatant from each well was discarded and replaced with 100μL of DMSO to dissolve the formazan crystals formed during MTT incubation. Absorbance (A_i, where i is the drug concentration) was measured via spectrophotometer at 570nm (Synergy H4, Agilent Technologies, Inc.). Relative viability was calculated using the following formula: relative viability = (A_i – A_background)/A_0.0 * 100%, where A_background is the absorbance from DMSO. IC₅₀ values were calculated by using a 4-parameter log-logistic model determined by the drm() function within the drc package in R. Here, the upper limit of the log-logistic model was set to 100%.