Figure 6.

Increasing activity of CBS in its reduced state increases the H₂S signaling in live HEK293 cells. A, HEK293 cells were exposed to 0.5 mm DTT or 0.5 mm oxidized GSSG-EE for 10 min. The cell lysates (50 μg) were resolved on SDS-PAGE and immunoblotted for CBS. B, H₂S production was measured in HEK293 cells using the methylene blue method. The inhibitors 1 mm AOAA and 2 mm dl-propargylglycine (PAG) were used to inhibit CBS and CSE, respectively. The data are presented as the means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001 versus vehicle. C, HEK293 cells were incubated with 2 μm SF7-AM for 30 min at 37 °C, washed, and then imaged using confocal microscopy. D, the same dishes of cells shown in C were incubated with 0.5 mm DTT for 30 min at 37 °C and then imaged. E, brightfield images of the same dishes of cells shown in D. Scale bar, 25 μm. F, HEK293 cells were incubated with 0.5 mm AOAA for 24 h to inhibit CBS activity and were then washed. Subsequently, the cells were incubated with 2 μm SF7-AM for 30 min at 37 °C, washed, and then imaged. G, The same dishes of cells shown in F were incubated with 0.5 mm DTT for 30 min at 37 °C and then imaged. H, brightfield images of the same dishes of cells shown in G. Scale bar, 25 μm. I, quantification of the confocal fluorescence images of H₂S signaling in HEK293 cells, with data from C, D, F, and G for comparison. The graph represents the relative fluorescence intensity compared with that of non-treated cells (C) and shows the means ± S.D. (n = 3). *, p < 0.05; N.S., not significant.