Nilotinib

Human ESCC cell lines KYSE150 and TE-1 were purchased from the Cell Bank at the Chinese Academy of Science (Shanghai, China). Cells were cultured in RPMI1640 medium, supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) and 1% penicillin-streptomycin (NCM Biotech, Soochow, China). All cell cultures were incubated at 37°C and 5% CO₂ in a humidified atmosphere. Nilotinib was purchased from MedChemExpress Co., Ltd. (Shanghai, China), while teniposide and ILK-IN-3 were acquired from Target Molecule Corp. (Shanghai, China). All compounds were dissolved in DMSO (MCE, Shanghai, China) before use.

Jump In T-Rex human embryonic kidney 293 cells (HEK293-JumpIn) with doxycycline-inducible expression of human OCT1, OCT2 or OCT3 (HEK293-JI-OCT1-3) were provided by Research Center for Molecular Medicine, Medical University of Vienna, Austria (CEMM) and constructed as reported previously [17 (link)]. HeLa cells were obtained from AddexBio (C0008001, San Diego, CA, USA). Corticosterone, 1-Ethyl-2-[(1-ethyl-2(1H)-quinolinylidene)methyl]quinolinium iodide (decynium-22), 1-Methyl-4-phenylpyridinium iodide (MPP⁺), 4-(4-diethylaminostyryl)-1-methyl-pyridinium iodide (DiASP), doxycycline hyclate, lansoprazole, ouabain, Dulbecco’s modified Eagles medium (DMEM), Minimum Essential Medium Eagle (MEM) and G418 were purchased from Sigma Aldrich (Merck, Darmstadt, Germany). Nilotinib, and ibrutinib were purchased from MedChemExpress (South Brunswick, NJ, USA). Hank’s Balanced Salt Solution (HBSS) and poly-D-lysine were obtained from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were bought from standard commercial resources and were of analytical grade.

Imatinib (17 mM stock; Sigma-Aldrich) was dissolved in water. Asciminib, nilotinib (20 mM and 10 mM stocks, respectively; MedChemExpress), dasatinib, S63845 (10 mM stocks; Selleckchem), and Z-VAD-FMK (20 mM stock; Calbiochem) were dissolved in dimethyl sulfoxide (DMSO). DMSO concentration was equalized in each well of a particular experiment.

Sorafenib, dasatinib, and nilotinib were purchased from MedChemExpress (MCE) (Monmouth Junction, NJ, USA). Material used in tissue culture, such as 100-mm, 6-well, and T-75 culture plates, were obtained from Fisher Scientific (Milford, MA, USA). The methyl thiazolyl tetrazolium (MTT) assay was procured from UFC Biotechnology (Buffalo, NY, USA). Flow cytometry kits using propidium iodide (PI) and annexin V staining were obtained from ThermoFisher (Waltham, MA, USA). Primers of p53, BAX, BCL-2, TNF-α, IL-6, IL-1β, and GAPDH as an internal housekeeping gene were procured from Integrated DNA Technologies (Leuven, Belgium).

K562 cells (CRL‐3343; American Type Culture Collection) were maintained in RPMI culture medium supplemented with 10% fetal calf serum (FCS), 2 mM Glutamine, and 1% penicillin–streptomycin (Life Technologies). They were tested for mycoplasma contamination and authenticated by STR profiling. Cells were maintained and subcultured before confluence every 72 h. For differentiation, cells were treated with 50 μM hemin (#16009‐13‐5, Sigma‐Aldrich), prepared as previously described (Smith et al, 2000 (link)), in the presence of 0.1% DMSO alone or with the caspase‐1 inhibitor VX‐765 (100 μM), the tyrosine kinase inhibitor nilotinib (0.1 μM), the RNA polymerase I inhibitor CX‐5461 (200 nM, 1 μM and 10 μM) (all from MedChemExpress). Cells were collected at different time points (0, 24, 48 h post‐hemin addition), centrifuged, washed twice with PBS, and stored at −80°C for further analysis.