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).