quence of HKI and HKII binding to the mitochondria is the prevention of cell death, possibly through interactions with the voltage-dependent anion channel and inhibition of the mitochon- drial permeability transition pore, which is a pro-death channel [24,25]. Overexpression of HKI and HKII in tissue culture protects against cell death [26], and both the mitochondrial- binding and glucose-phosphorylation activities of these proteins are required for their protective effects [27]. The regulation of HKIII and its potential role in cytoprotection are not well characterized. In this report, we studied the regulation of HKIII at the transcriptional and protein levels as well as its role in cellular protection. We show that of the various factors that regulate HKII, only hypoxia influences HKIII levels through a HIF-dependent pathway. Mutations in the predicted N-terminal glucose and G6P binding sites of HKIII decreased activity in the C-terminus, which suggests that the two halves of HKIII functionally interact. HKIII also protects against cell death, and its overexpression increases ATP levels, decreases the oxidant- induced production of reactive oxygen species (ROS), attenuates the oxidant-induced reduction in mitochondrial membrane potential, and increases mitochondrial biogenesis. To better characterize the importance of mitochondrial binding on the protective effects of HKs, we attempted to target HKIII to the mitochondria by replacing the N-terminal 32-amino-acid region of HKIII with the mitochondrial binding domain of HKII. However, the resultant chimeric protein aggregated, suggesting that unlike HKI and HKII, the N-terminal region of HKIII may play a role in tertiary structure formation and in maintaining protein solubility. Methods Cell culture N1S1 cells are descended from the Novikoff rat hepatoma cell line and were a gift from Dr. Andrew Larson (Northwestern); the cells were maintained as previously described [28]. HEK293 (HEK) cells were obtained from the American Type Culture Collection (ATCC) and maintained in MEM (Cellgro, Mediatech Inc.) supplemented with 10% fetal bovine serum (Cellgro, Mediatech, Inc.); sodium pyruvate; and penn/strep (Gibco, Invitrogen Corporation). Cell treatments For the hypoxia studies, cells were incubated at 1.5% O2 for 24 hours. To stabilize HIF isoforms under normoxic conditions, cells were incubated with 100 or 300 mM of the prolyl hydroxylase inhibitor Dimethyloxalyl glycine (DMOG) (Roche) for 24 h before isolation of mRNA and protein. For evaluation of cAMP or AMP- activated kinase (AMPK) activation, N1S1 cells were incubated for 24 hours in serum free media with 5 mM forskolin or 1 mM aminoimidazole carboxamide ribonucleotide (AICAR), respective- ly (Sigma). RNA isolation and Western blotting Total RNA from cells was extracted with RNA STAT (Tel-test Inc) and reverse transcribed with the TaqMan preamplification system (Applied Biosystems). Real time polymerase chain reaction (PCR) was performed using SYBR Green and assayed on the ABI 7500 Detector (Applied Biosystems). Primer sequences for all targets are listed in Supplemental Table S1. All target mRNA levels were normalized to an interal housekeeping control (18S or b2M). For Western blot, cells were lysed and protein samples were run on an SDS PAGE gel. Western blots were performed using HK3 Regulation and Protection commercially available antibodys for HKIII and actin (Abcam, and Santa Cruz respectively). HKIII and HIF constructs A plasmid containing HKIII cDNA was kindly provided by Dr. Graeme Bell (University of Chicago). 59 EcoR1 and 39 BamH1 sites were engineered into the sequence around the region of interest, then DNA was amplified via PCR and gel purified. The construct was cloned into the TOPO vector (Invitrogen), and the DNA sequence was confirmed. Using this construct as a template, we introduced ATG codons into PCR primers that initiated protein translation at amino-acid positions 9, 17, 25, and 33, thereby generating truncated versions of the HKIII protein lacking the first 8, 16, 24, and 32 residues of the native sequence, respectively. To generate a chimeric HKIII protein containing the HKII mitochondrial-targeting sequence, the construct lacking the first 32 amino acids was used as a template with primers encoding the 21-amino-acid N-terminal region of HKII. After all DNA sequences were confirmed, the HKIII fragments were excised via restriction digestion and subcloned into the pEGFP-N3 vector (Clontech). Constitutively active HIF-1a and HIF-2a constructs have double proline-to- alanine point mutations preventing hydroxylation and subsequent degradation [29]. These constructs were generously donated by Dr. Mircea Ivan (Indiana University). Viability assays The HKIII-GFP fusion construct or the GFP vector was transfected into HEK using Lipofectamine2000 (Invitrogen). Expression was confirmed with fluorescent microscopy, and Western blot analyses of lysates from transfected cells as previously described [27]. Experiments were performed 36 to 48 hours after transfection, and cells were cultured overnight in basal MEM without supplements before each experiment. To induce death, cells were exposed to exogenous oxidative stress using H2O2 at the dose and time points indicated. Cell viability was assessed using propidium iodide exclusion and Annexin V conjugated to Alexa- 350 (Invitrogen) according to the manufacturer’s instructions. Results were quantified via flow cytometry using the BDFACS LSR flowcytometer and analyzed using the FlowJo software program. ATP assessment ATP levels were determined using the Cell Titer Glo Assay from Promega, according to the manufacturer’s recommenda- tions. Luminescence was recorded with the SpectraMax Gemini (Molecular Devices), and measurements were normalized to the total number of viable cells in each sample, as determined by trypan blue exclusion with cell counts made on a hemocytometer. ROS measurements ROS levels were measured using the MitoSOX fluorescent marker (Invitrogen), which selectively reacts with superoxide. Briefly, the MitoSox probe (1 mM) was added to cultured cells 10 min prior to the end of the indicated treatment condition. Cells were then washed with PBS and incubated for 5 min in MEM containing the Hoechst33342 nuclear marker (Invitrogen). The media was replaced with fresh MEM and ROS levels were evaluated via fluorescent microscopy. Images from 5 representa- tive fields were captured for each individual sample, and fluorescent intensity in the 510/580 nm spectra was evaluated using the ImageJ software program (NIH), and normalized to cell number. PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13823