Hdmec

The pharynx carcinoma-derived FaDu epithelial cell line and the lung adenocarcinoma-derived Calu-3 epithelial cell line were obtained from the American Type Culture Collection. HDMEC (Primary Human Dermal Microvascular Endothelial Cells) were purchased from Promocell. FaDu cell lines were grown in Ham F-12 medium (PAA Laboratories) supplemented with 10% fetal calf serum (FCS; PAA Laboratories), 20 mM HEPES (PAA Laboratories) and 1% penicillin-streptomycin-amphotericin (PSA; PAA Laboratories). Calu-3 cell lines were grown in Opti-MEM (Gibco) supplemented with 5% fetal calf serum (FCS; PAA Laboratories). HDMEC were grown in ECM (Endothelial Cell Medium with supplements provided by the manufacturer, Promocell), 20 mM HEPES (PAA Laboratories) and 1% penicillin-streptomycin-amphotericin (PSA; PAA Laboratories). Cells were grown at 37°C in a humidified incubator under 5% CO₂.
The cells were fixed using a solution of PBS-4% paraformaldehyde (PFA) during 20 min.

HDMECs (PromoCell) were purchased and cultured in endothelial cell growth medium MV (PromoCell) at 37 °C in 5% CO₂ until reaching 70–80% confluency. HDMECs were collected with the DetachKit (Promocell) and plated on the insert plates of Millicell-96 transwell (Merck Millipore, Darmstadt, Germany) at 5 × 10⁴ cells in 75 μl medium/well. Two hundred and fifty microliters of medium was added to the receiver plate and incubated for 2 days at 37 °C in 5% CO₂. The insert plates were then replaced to the new receiver plate. The medium was aspirated from the insert plates, and new medium with imatinib, asciminib, or GNF-2 was added at 50 μl/well at various concentrations as indicated in each figure, and incubated for 3 h. Asciminib and GNF-2 were synthesized in Mitsubishi Tanabe Pharma Corporation. Subsequently, 10 μl of FITC-conjugated human IgG at 187.5 μg ml⁻¹ (Jackson ImmunoResearch) was added to the insert plates, and incubated for 30 min. Ten microliters of thrombin was added at 5 U ml⁻¹ and incubated for another 60 min. Finally, the insert plates were removed, and fluorescence intensity of the FITC-conjugated human IgG in the receiver plates was measured by EnVision (PerkinElmer, Waltham, MA).