Cells were seeded overnight and incubated with colchicine (10 nM), paclitaxel (10 nM), and CH-2-77 (2.5 nM, 5 nM, and 10 nM) for 24 h. Then, cells were digested with trypsin-EDTA (#2520056, Gibco) and stained with Annexin-V-FITC (eBioscience, 5 μL) and propidium iodide (PI, 10 μL) in Annexin-V-FITC binding buffer (100 μL) for 10 min. Apoptotic cells were detected by flow cytometry. For cell cycle distribution, cells with the same treatments as the apoptosis assay were harvested, washed, and fixed. After permeabilization, cells were stained with rabbit anti-phospho-histone H3 (Ser 10) antibody (#9701, 1:50, CST) for 1 h at room temperature followed by a 30 min incubation with Alexa Fluor 488 goat anti-rabbit secondary antibody (# A-11008, 1:50, Molecular Probes). After washing 2 times with PBS, cells were resuspended in PI/RNase staining solution and detected by cytometry after a 5 min incubation. Cells in sub-G1, G1, S, and G2 phases were gated by a reported method (18 (link)). The experiment was conducted using three biological replicates with three technical replicates per treatment per cell line.