HK3 Regulation and Protection Mitochondrial Membrane Potential Mitochondrial membrane potential (Dym), was evaluated using the fluorescent marker tetramethylrhodamine ethyl ester TMRE (Sigma) as previously described [27]. Briefly, cells were loaded with TMRE 10 min prior to the start of the indicated treatment. Fluorescent microscopy images were taken to demonstrate relative changes in TMRE intensity. Results were quantified via flow cytometry as describe above. Figure 1. Hypoxia regulates HKIII expression in N1S1 cells via HIF signal transduction. (A) HKIII mRNA and protein levels (left and right panels, respectively) were measured in N1S1 cells cultured under normoxic or hypoxic (1.5% O2) conditions for 24 hours. HKIII levels were significantly higher after hypoxia treatment. (B) N1S1 cells were cultured under normoxic conditions for 24 hours with or without a HIF stabilizer (DMOG, 100 and 300 mM). HKIII mRNA and protein levels increased significantly after DMOG treatment. (C) HKIII mRNA levels were significantly increased in N1S1 cells expressing constitutively active (ca) HIF-1a or HIF2a constructs under normoxic conditions, 24 h after transfection. (D) HKIII mRNA levels were significantly increased after hypoxia in another rat hepatoma cell line (McA-RH 7777), as well as a human myeloid leukemia cell line (HL 60). This response was not observed in HEK293 cell which had no detectable HKIII expression under normoxic or hypoxic conditions. *P,0.05 v. control; Data are presented as mean6SEM, N = 3–5 for all groups. doi:10.1371/journal.pone.0013823.g001 PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e13823