Colony formation assay
For colony formation assay, KYSE150 and KYSE510 cells were plated in
6-well plates (1×103 cells/well) and cultured under
standard culture conditions overnight. Then, the medium was replaced
with gradient concentrations of ixazomib and vehicle for 2 weeks until
the colonies were visible in the culture plate. The supernatants were
discarded and washed with PBS twice, 4% paraformaldehyde used to fix
the colonies, and stained with 0.1% crystal violet solution for 30
minutes. Finally, the excess staining solution was washed away with
distilled water and photographed on a microscope and used Image J
software to calculate the number of clones.