2.6. Apoptosis assay
The Cell Death Detection ELISAplus kit (Roche) was used to measure apoptosis. HUVEC were seeded in 96-well plates at 30,000 MK5108 per well. Twelve hours later, cells were either treated with DMSO as a control or treated with NVP-BEZ235 (100 nM), PP242 (100 nM), rapamycin (10 nM) in combination or not with UO126 (10 μM) for 48 h. Subsequently cells were harvested and apoptosis was determined following the manufacturer’s instructions. Results are represented as the mean enrichment factor (absorbance of the treated cells/absorbance of the control cells).
2.7. Tubulogenesis
HUVEC were treated with rapamycin, NVP-BEZ235, PP242, UO126, or a phycoerythrin combination of UO126 with mTOR inhibitors, or DMSO as a control for 4 h. HUVEC were subsequently harvested and cultured in matrigel-precoated 96-well plate for 6 h at 37 °C (1 × 104/well). Tubulogenesis was visualized with an Olympus inverted microscope and the numbers of branching points were counted. Points generating at least three tubules were counted.
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