Fig. 1. Dose- and time-dependent effect of H₂O₂ on superoxide production in K562 cells and K562/NOX5 cells.

K562 and K562/NOX5 cells were assayed for superoxide production using the Diogenes reagent and for NOX5 protein by immunoblot. (A) Immunoblot of crude membranes using antibodies to NOX5 and protein disulfide isomerase (PDI; loading control). (B, D) Chemiluminescence from cells incubated with (broken line) or without (solid line) 100 μM H₂O₂ for the indicated times. (C, E) Superoxide production was measured in cells incubated for 10 minutes with various concentrations of H₂O₂ (0–500 μM). After normalization by subtraction of the zero-time value of the chemiluminescence output, the area under the curve was calculated as a measure of total superoxide production. (F) Superoxide production was determined in whole K562/NOX5 cells incubated with or without 100 μM H₂O₂ and/or catalase (900 U). After normalization as above, the superoxide production was expressed as a percent of non-treated cells without H₂O₂ (NT). The data expressed are the means +/− SEM of 3 to 6 independent experiments. Asterisks indicate statistical significance versus control cells without H₂O₂: * P<0.05, ** P<0.01.