Figure 1.

Glutathione depletion by buthionine sulfoximine inhibits the potentiating effects of Nrf2 activators on 1,25D₃-induced differentiation and elevates ROS levels in HL60 cells. Cells were preincubated with vehicle (water) or 30 μM BSO for 24 h, followed by treatment with the indicated concentrations of 1,25D₃, carnosic acid (CA), or monomethyl fumarate (MMF), alone or in combination, for another 48 h. (a) Representative flow cytometric data showing the enhancing effect of CA on 1,25D₃-induced surface expression of CD14 and CD11b and the inhibitory effect of BSO on this enhancement. (b) Summarized CD14 and CD11b expression data, as exemplified in panel (a). (c) Changes in the total glutathione content, as determined by the glutathione reductase recycling assay following 24 h of preincubation with BSO followed by 16 h of treatment with CA or MMF. (d) Averaged ROS levels measured as DCF geometric mean fluorescence intensity (MFI) units (% of control). Cells were preincubated with BSO for 24 h and treated with or without CA or MMF for an additional 48 h. The data are means ± SD of at least 3 independent experiments performed in duplicate. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 vs. untreated control group; ##, p < 0.01; ###, p < 0.001 vs. sum of the effects of single agents; $$, p < 0.01; $$$, p < 0.001; $$$$, p < 0.0001 vs. corresponding BSO-untreated group.