HK3 Regulation and Protection Figure 6. HKIII overexpression preserves mitochondrial membrane potential in cells exposed to oxidant stress. Mitochondrial membrane potential was assessed in HEK cells overexpressing HKIII or GFP after treatment with H2O2, using the fluorescent dye tetramethylrhodamine ethyl ester TMRE (orange). (A) Fluorescent images taken 3 and 6 h after exposure to 500 mM of H2O2 demonstrate a pronounced reduction in TMRE fluorescence over time in cells overexpressing GFP that was attenuated in (C) cells overexpressing HKIII. TMRE images are overlayed with the Hoechst nuclear stain (blue) for (B) GFP and (D) HKIII. The fluorescent GFP overlay for each image is presented in the supplemental material. (E) TMRE levels, quantified by flow cytometry, were significantly higher in cells overexpressing HKIII v GFP control 3 and 6 h after treatment with 500 mM of H2O2. N = 3–6. *P,0.05 vs GFP. Data are presented as mean6SEM. doi:10.1371/journal.pone.0013823.g006 PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e13823