Figure 1. Hcy suppresses cell viability, induces pyroptosis/apoptosis in ECs. HUVECs were cultured to 80% confluence, synchronized (serum free, 6hr), and then switched to 0.5%FBS medium containing L-Hcy, L-Cys, LPS, and H₂O₂ (500µM) for 24hr as indicated.

A, Pyroptosis and apoptosis gating (FCM, AV/7AAD/Casp staining). Cells were treated and stained by AnnexinV-Pacific Blue and 7AAD, and Casp1/9 activity kit for FCM analysis (dot plot shown in online Figure II). Necrotic cells were excluded from intact cells by their content of nuclear debris. Casp9 activity was used as a marker of apoptosis. Q2+Q3 cells were define as apoptosis. Q4 was defined as pyroptosis. B, Cell viability. Cells in 96-well plate were fixed, stained by crystal violet for cell viability. C, Hcy/LPS induced Pyroptosis/apoptosis (dot plot shown in online Figure IV). Cell death forms were identified by FCM using Annexin V-FITC/PI staining (dot plot shown in online Figure II). D, DNA fragmentation. DNA fragmentation was determined by TUNEL staining (images shown in online Figure V). Data are representative of 4 separated experiments and presented as Mean±SEM. Value on the top of bars are normalized by the mean of the control or Q1. *, P<0.05 vs control; #, P<0.05 vs LPS alone in same group; $, P<0.05 vs same dose of Hcy alone; Syn is synergy effect. £, p<0.05. §, p<0.05 vs Q4. FCM, flow cytometry.