Cell cycle analysis
For cell cycle analysis, we performed using the Cell Cycle Kit
(Beyotime, China). KYSE150 and KYSE510 cells were treated with vehicle
(0.1% DMSO) or ixazomib (0.25, 0.5, 1 μM) for 24 h. Then these cells
were washed by PBS (0.1M, pH 7.4) three times, and fixed in 70% ethanol
solution at -20℃ overnight. The fixed cells were washed with cold PBS to
remove excess ethanol solution and stained with propidium iodide (PI)
staining buffer. Samples were incubated at 37℃ for half an hour and
using BD Accuri™ C6 (BD Bioscience, USA) for assay. The experiments were
repeated three times, and data were analyzed with ModFit LT software
(Verity Software House, Topsham, ME).