In the apoptosis assay, HUVECs (1.5 × 105 cells/well) were plated onto six-well plates and were treated with decreasing dilutions (1:2500-1:250) of A009 or HT for 24h (A) and 48h (B) in complete M199 medium. Vehicle (ethanol) and M199 medium alone were used as negative and positive controls, respectively. Fluorescence signal for Annexin V and 7 AAD (7- amino-actinomycin D) was used to detect viable, dead or apoptotic cells; the fluorescence signal was measured with a FACSCanto flow cytometer and data were analyzed with the FACSDiva Software 6.1.2. After 24h of treatment with A009, HUVECs underwent apoptosis in a dose-dependent manner, while HT induced apoptosis only at the highest concentration (1:250). Mean ± SD of 3 independent experiments is shown. Student' s t-test was used to determine p-value. (*p<0.05 **p<0.01)
