Kin193

Cell proliferation was determined using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), according to the manufacturer’s instructions. For the CCK-8 assay, cells were seeded in 96-well plates at 2.0×10³ cells per well in RPMI-1640 medium with 10% fetal bovine serum. After a 24 h incubation, cells were treated with KIN-193 (MedChemexpress, Monmouth Junction, NJ, USA) at various concentrations (0, 5, 10, 15, 20, 25, and 30 µM) and incubated in complete medium for 24, 48, or 72 h. Absorbance was measured at 450 nm using a microplate reader (Perkin Elmer, Waltham, MA, USA) at the specified time points (24, 48, or 72 h). For colony formation assays, cells were seeded into 6-well plates (250 cells/well). After incubation for 24 h, cells were treated with various concentrations of KIN-193 (0, 10, 20, and 30 µM) and incubated in complete medium for 12 days. Plates were washed with phosphate buffered saline (PBS; Invitrogen Life Technologies, Carlsbad, CA, USA) and stained with 0.1% Crystal Violet. The number of colonies consisting of >50 cells were calculated. All compounds were dissolved in PBS.