HK3 Regulation and Protection Figure 5. Overexpression of HKIII increases ATP levels and reduces oxidant-induced ROS levels. (A) ATP levels were significantly higher in cells overexpressing HKIII both at baseline and after treatment with exogenous H2O2 (500 mM, 6 h). Cells were incubated overnight in serum free basal MEM media, and ATP levels were determined via the Cell Titer Glo assay, with luminescence (RLU) normalized to the number of viable cells. N = 6. (B) HEK293 cells were transfected with HKIII-GFP or GFP, treated with or without 500 mM H2O2 (3 h), and then ROS levels were measured with a fluorescent marker of superoxide production (MitoSox, red). Nuclei are labeled with Hoechst (blue), and the GFP overlay for these images is presented in the supplemental material. (C) HKIII overexpression was associated with significantly lower ROS levels in H2O2-treated cells. (N = 3, ROS levels for each sample represent the average fluorescent intensity from 5 separate fields, each normalized to cell number). *P,0.05 vs GFP. Data are presented as mean6SEM. doi:10.1371/journal.pone.0013823.g005 PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e13823