Cell apoptosis assay
For simultaneous measurement of cell apoptosis, we performed using the Annexin V–FITC/PI apoptosis kit (Beyotime, China) according to the manufacturer’s instruction. KYSE150 and KYSE510 cells were treated with vehicle (0.1% DMSO) or ixazomib (0.25, 0.5, 1 μM) for 24 h. Then the cells were harvested and washed twice with cold PBS, and Annexin binding buffer (500 μl) was added to each sample. Thereafter, cells were stained by Annexin V–FITC and PI solution in the dark for 15 min. The stained cells were analyzed by flow cytometry (Novocyte, ACEA Biosciences) at the fluorescence emission of 530 nm.