Egmtm 2 mv microvascular endothelial cell growth medium 2 bulletkit

HUVEC were detached and resuspended in EGM^TM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM (Lonza, CC-3202) to a concentration of 2 × 10⁵ cell/ml. NPC/TPC/fibroblast/smooth muscle cells or HK2/ROCK1/ROCK2 stably depleted NSCLC/HCC-derived TPC/ROCK2 overexpressing HK2 stably depleted TPC/scramble transfected TPC were first labeled with 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (TONBO, 13-0850), which were then co-cultured with HUVEC at ratio of 1:10 (NPC/TPC/fibroblast/smooth muscle cells vs HUVEC) on matrigel (R&D, 3432-010). In all, 20,000 HUVEC per well were seeded onto a 96-well plate. For the rescue experiments, TPC were first pre-treated with 200 μM 3-BP or 0.4 μM GSK429286A/HA-1077 inhibitors at room temperature for 15 min. The inhibitors were then removed by centrifuging. For the conditioned medium experiment, NPC were first pre-treated with conditioned medium from A549 or HepG2 cells for 6 h. These cells were then co-cultured with HUVEC as described above. Images were taken by using a fluorescence microscopy 24 h after co-culturing (Nikon, Ni-U). The total tube length, branch points, and number were measured in each group.

HMVEC-d cells (catalog #: CC-2543 Lonza, Basel, Switzerland) were propagated (passages 4–8) and maintained at 37 °C in humidified air with 5% CO₂ in EGM^TM 2MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM (catalog #: CC-3202, Lonza, Basel, Switzerland) supplemented with growing factors, antibiotics, and 10% fetal bovine serum according to the manufacturer’s specifications.

Hec50 cells were kindly provided by Dr. Erlio Gurpide (New York, NY, USA) [18 (link)] and grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco Corporation, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum. KLE cells were purchased from ATCC and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. Human umbilical vascular endothelial cells (HUVEC) were purchased from ATCC and grown in EGMTM -2 MV Microvascular Endothelial Cell Growth Medium (2 BulletKit, Lonza, Alpharetta, GA, USA). To ensure rigor and reproducibility, the identity of all cell lines was confirmed using the CODIS genotyping test (Cat. No. CL1003, Bio-Synthesis, Lewisville, TX, USA).