Ki8751

Human glioblastoma multiforme cancer cell line U87 was purchased from the American Type Culture Collection (ATCC; Wesel, Germany), and U38 cells were characterized in Professor Monica Nister laboratory at Karolinska Institutet [20 (link), 21 (link)]. The cell lines have been authenticated using Short Tandem Repeat (STR) profiling within the last three years. U87 cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, heat inactivated) and 1% penicillin–streptomycin at 37 °C with 5% CO₂. U38 cells were cultured using Minimum Essential Medium (MEM; Gibco) containing 10% FBS and 1% penicillin–streptomycin at 37 °C with 5% CO₂. Briefly, the U87 and U38 cells were seeded in 6-well plates at the density of 1.5 × 10⁵ cells per well. After overnight attachment, the cell media were replaced by DMEM or MEM containing vehicle (0.01% dimethyl sulfoxide) or Ki8751 (Tocris Bioscience, Bristol, UK). The cells were then cultured for 24, 48 or 72 h before further experiments. All experiments were performed with mycoplasma-free cells.

In matrigel-coated 96-wells, 5×10⁴ HUVEC cells were layered in EC medium supplemented with vehicle, 50ng/ml of recombinant human VEGF-A (rhVEGF-A, Sino Biological Inc., North Wales, PA, USA), or with 10μl of MSC-reconditioned or not IRIS291 or IRIS293 CM concentered 10 times using centricon (30K). Experiments were done in the presence or absence of 10nM of the VEGFR2 inhibitor “Ki8751” (10nM, TOCRIS Bioscience, Bristol, UK). After 6h of culture growth at 37°C images of tube formation were captured under light microscope. Experiments were done in triplicates performed 3 separate times.