Figure 2. Hcy activates endothelial casp1 prior to casp8,9,3 activations in the presence/absence of LPS.

HUVECs were cultured and treated with L-Hcy (500µM) and/or LPS (10 µg/mL) as described in Fig. 1. Casp1,8,9,3 activities were measured by using a manufacture’s kit and Western Blotting respectively. Caspase activated cells were FAM positive and labeled by green fluorescence (images shown in online Figure VI). A, Casps Activity (24hr). Casp1,8,9,3 activities were detected by fluorescence spectrometry through a 0~24hr time-course. See supplement Table 1 for values and statistical analysis. B, Casp1 activity (FCM, 2 & 24Hr). Casp1 activity was examined after 2hr and 24hr treatment by FCM. C, Casp8,9,3 activities (FCM, 24 Hr). Casp8,9,3 activities were detected by FCM. D, Casp1 protein and activity. A 20kDa cleavage fragment indicated activated Casp1 and detected by WB, after 2hr and 24hr L-Hcy incubation, with or without LPS presence. E, Schematic sketch show casp1 activation and detection by WB, Fluorometer or FCM. *, p<0.05 vs control; £, p<0.05 vs L-Hcy treatment alone. In A, ‡, p<0.05 vs Casp1 activity at 0hr; ‖, p<0.05 vs Casp8 activity at 0hr; ╫, p<0.05 vs Casp9 activity at 0hr; §, p<0.05 vs Casp3 activity at 0hr. Values are Mean±SEM; n=4.