Cultured cells were assayed for viability using PrestoBlue HS (Thermo Fisher Scientific) based on manufacturer’s instructions. Briefly, FLC cells were cultured in 96 well plates in RPMI medium supplemented with 10% FBS and 2% Penicillin/Streptomycin. Huh7-Chimera cells were cultured in 96 well plates with DMEM medium supplemented with 10% FBS and 2% Penicillin/Streptomycin. Huh7-Chimera cells were seeded at 5000 cells/well and growth measured daily. For experiments with doxycycline, 100 ng/ml of doxycycline was added for the indicated time periods before PrestoBlue readout. PrestoBlue was added at 1:10 and cells were incubated for 1–2 hours before plate reader fluorescence measurement at 560 nm excitation and 590 nm emission in a Spark Tecan instrument.
For experiments with Huh7-Chimera cells, raw absorbance values were used and normalized by wells not containing cells. For experiments with FLC cells, viability was calculated compared to cells treated with 20 µM chaetocin (Positive, SelleckChem S8068) and untreated (Negative) as percent survival = (Positive-Treated)/(Positive-Negative) * 100.
For experiments with Huh7-Chimera cells, raw absorbance values were used and normalized by wells not containing cells. For experiments with FLC cells, viability was calculated compared to cells treated with 20 µM chaetocin (Positive, SelleckChem S8068) and untreated (Negative) as percent survival = (Positive-Treated)/(Positive-Negative) * 100.