Ponatinib

For our experiments, HCAEC line, cell culturing medium, trypsin-EDTA, Hank’s balanced salt solution (HBSS) and dimethyl-sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Plastic cell culturing flasks and plates were purchased from Greiner Bio-One (Nürtingen, Germany). Ponatinib was ordered from Cayman Chemical (Ann Arbor, MI, USA). HCAECs were cultured in MesoEndo growth medium in 75 cm² flasks at 5% CO₂ and 80% humidity. When the cells reached 80–90% confluence in the flask, they were plated in 6-well culture plates (10⁵ cells/well), and after 2 days, the cells were treated for up to 48 h with clinically relevant and supratherapeutic concentrations of Ponatinib. The final concentrations of Ponatinib were 50, 150, and 1000 nM, DMSO (0.2 v/v %) was used as a negative control, and tumor necrosis factor alpha (TNF-α) (100 ng/mL; Gibco, Waltham, MA, USA) was used as a positive control. After the treatments, the cells were collected from both the plate and the culture medium.

All cell lines were purchased from DSMZ (Braunschweig, Germany) or ATCC (Manassas, VA, USA). The cell lines were regularly tested to exclude mycoplasma contamination and authenticated. All cell lines were grown at 37 °C and 5% CO₂. A549 cells were cultured in DMEM medium (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA). All other cell lines were cultured in RPMI 1640 medium (Gibco™, Thermo Fisher Scientific). Both media were supplemented with 10% fetal bovine serum (FBS), 10 U/mL penicillin, 10 µg/mL streptomycin and 2 mM L-glutamine (all Gibco™, Thermo Fisher Scientific). Bortezomib (S1013; Selleck Chemicals, Houston, TX, USA), cabozantinib (S1119; Selleck Chemicals), cytarabine (PHR1787; Millipore Sigma, Burlington, MA, USA), gilteritinib (HY-12432; MedChemExpress, Monmouth Junction, NJ, USA), ispinesib (HY-50759; MedChemExpress), midostaurin (M1323; Millipore Sigma), ponatinib (11494; Cayman Chemical, Ann Arbor, MI, USA), quizartinib (17986; Cayman Chemical), venetoclax (HY-15531; MedChemExpress) and WS6 (S7442; Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO; Carl Roth, Karlsruhe, Germany) and diluted further in culture medium immediately before use.