Cell cytotoxicity assay

Previous reports indicated that 50% of HCT-8 cells were viable (IC₅₀) following administration of FK228 at concentrations of 29.46 nM [30]. We performed cytotoxicity assays on HCT-116 cells using varying concentrations of FK228 according to previously described methods [30]. When HCT-116 cells reached 75% confluence, a cell proliferation and cytotoxicity assay was performed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to manufacturer instructions. Briefly, a 96-well plate was pre-incubated for 24 h, followed by addition of different concentrations of FK228 (Sigma-Aldrich, St. Louis, MO, USA) to each well. CCK-8 solution (10 μL) was added and incubated for an additional 1 to 4 h. Cell viability was determined by measuring the absorbance at 450 nm. Cell cytotoxicity was calculated using the HTC-116 cell-viability measurements following treatment with FK228 The FK228 concentration resulting in 50% cell viability (IC₅₀) was used as the fixed working concentration for later experiments.