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.