Lung cancer cells seeded in 96-well plates (2500 cells/well) were dosed 24 h later with different inhibitors for 72 h. Cell viability was determined by PrestoBlue (PB) Cell Viability Reagent (ThermoFisher Scientific) by following the manufacturer’s instructions [14 (link), 31 ]. The PB reagent was added into media directly (1:10 dilution) and incubated for 30 min-2 h and then the fluorescence was read (excitation 570 nm; emission 600 nm) at recommended time of incubation. The efficacy of drugs on cell growth was normalized to untreated control. Each data point was generated in triplicate and each experiment was done three times (n = 3). Best-fit curve was generated in GraphPad Prism [(log (inhibitor) vs response (−variable slope four parameters)]. Error bars are mean ± SD. The combination index (CI) was calculated by ComboSyn software (ComboSyn Inc., http://www.combosyn.com/ ).
Clonogenic assay was done as we described previously [14 (link), 31 –33 (link)]. In brief, cells seeded in 6-well plates (3000 cells/well) were dosed 24 h later and continually treated with rapamycin for 7 days (refresh drugs every 3 days), the resulting colonies were stained with crystal violet (0.5% dissolved in 25% methanol).
Clonogenic assay was done as we described previously [14 (link), 31 –33 (link)]. In brief, cells seeded in 6-well plates (3000 cells/well) were dosed 24 h later and continually treated with rapamycin for 7 days (refresh drugs every 3 days), the resulting colonies were stained with crystal violet (0.5% dissolved in 25% methanol).
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