A5955 100

HEK293 FT wild-type and APEX1-KO cells were plated into 96-well plates at a density of 1.3 × 10⁴ cells/well in DMEM with 10% fetal bovine serum (FBS) and antibiotic/antimycotic mix (Sigma, A5955-100, St. Louis, MI, USA). CH12F3 wild-type and APEX1-KO cells were collected by centrifugation and resuspended in phenol red-free RPMI-1640 with L-glutamine, 10% FBS, and antibiotic/antimycotic mix (Sigma, A5955-100, St. Louis, MI, USA) and then plated on 96-well plates at a density of 2.2 × 10⁴ cells/well. The plates were incubated for 24 h at 37 °C in a 5% CO₂ humidified incubator. Immediately prior to use, the Ape1 inhibitors and methylmethane sulfonate (MMS) were diluted with a culture medium containing 3% FBS. For both HEK293 FT and CH12F3 cells, the FBS in the medium was reduced from 10% to 3% before the addition of inhibitors or MMS. After 24 h of treatment, MTT (3-(4,5-dethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was then added at a concentration of 0.5 mg/mL, followed by a 3-h incubation. Stop solution (1% SDS and 0.01 M HCl) was used to dissolve formazan crystals, and the absorbance was measured at 590 nm.