Figure 6. Hcy/LPS-induced intracellular ROS levels determines cell destiny (pyroptosis/apoptosis/viable cell) in ECs.

HUVECs were cultured and treated with L-Hcy (500µM), LPS (10 µg/mL) or H₂O₂ (500µM) for 24hr as described in Fig. 1. Triple staining was applied to detect intracellular ROS (DHE) and apoptosis/pyroptosis simultaneously (Annexin-V-FITC and 7-AAD). A, ROS⁺/apoptosis and ROS⁺/pyroptosis cells (FCM). The ROS⁺/apoptosis and ROS⁺/pyroptosis cells were defined and quantified (dot blot and histogram shown in online Figure VIII). B, ROS level quantification in viable, pyroptotic, apoptotic cell population. ROS level was quantified by mean fluorescence intensity (MFI) of DHE in apoptotic (Annexin V⁺), pyroptotic (7-AAD⁺/Annexin V⁻), and viable (7-AAD⁻/Annexin V⁻) cells. C, ROS gradient and apoptosis/pyroptosis. Four regions of ROS level (low, intermediate, high, and extreme high) were defined by their DHE fluorescence intensities. Apoptosis/pyroptosis was quantified in each region. Numbers above each bar is the percentage normalized by the mean of control. *, p < 0.05 vs control in same group or same ROS gradient; #, p < 0.05 vs Hcy in same group or same ROS gradient; †, p < 0.05 vs same treatment in pyroptosis; $, p < 0.05 vs same treatment in viable cells; ‖, p < 0.05 vs same treatment in G1; ‡, p < 0.05 vs same treatment in G2; £, p < 0.05 vs same treatment in G3.) Values are Mean±SEM; n=4.