Ponatinib Effects on HCAEC Cultures

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.