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Cell Metab. Author manuscript; available in PMC 2011 Jul 22.
Published in final edited form as:
Cell Metab. 2005 Jun; 1(6): 393–399.
doi: 10.1016/j.cmet.2005.05.003
PMCID: PMC3141219
NIHMSID: NIHMS312152
PMID: 16054088

Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-α activation

Kyle D. Mansfield,1 Robert D. Guzy,2 Yi Pan,1,3 Regina M. Young,1,3 Timothy P. Cash,1 Paul T. Schumacker,2,4 and M. Celeste Simon1,3,*
Author information Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

Summary

While cellular responses to low oxygen (O2) or hypoxia have been studied extensively, the precise identity of mammalian cellular O2 sensors remains controversial. Using murine embryonic cells lacking cytochrome c, and therefore mitochondrial activity, we show that mitochondrial reactive oxygen species (mtROS) are essential for proper O2 sensing and subsequent HIF-1α and HIF-2α stabilization at 1.5% O2. In the absence of this signal, HIF-α subunits continue to be degraded. Furthermore, exogenous treatment with H2O2 or severe O2 deprivation is sufficient to stabilize HIF-α even in the absence of cytochrome c and functional mitochondria. These results provide genetic evidence indicating that mtROS act upstream of prolyl hydroxylases in regulating HIF-1α and HIF-2α in this O2 sensing pathway.

Keywords: hypoxia, reactive oxygen species, HIF-1α, HIF-2α, anoxia

Introduction

When confronted with a decrease in O2 levels, cells initiate a myriad of responses to combat the imposing stress. Many hypoxic transcriptional responses are mediated by the hypoxia inducible factors (HIFs) whose activity is regulated mainly through the stability of their α subunits, which are hydroxylated on prolines within their oxygen-dependent degradation domains (ODDs) under normoxic conditions (Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001). The von Hippel-Lindau tumor suppressor (pVHL) recognizes and targets hydroxylated HIF-α for degradation via the 26S proteosome. Hypoxia inhibits hydroxylation and stabilized HIF-α translocates to the nucleus, dimerizes with a HIF-β subunit (ARNT) and activates transcription (Bruick, 2003; Wenger, 2002). Because of their O2 dependence (McNeill et al., 2002) and relatively high Km for O2 in vitro (Hirsila et al., 2003), it has been proposed that the HIF-specific hydroxylases may directly sense O2 deprivation to stabilize HIF.

Mitochondria have also been implicated in multiple HIF-dependent and independent hypoxic responses through the hypoxic production of intracellular mtROS. These hypoxic mtROS potentially regulate many cellular processes including HIF-1α stability and transcriptional activity (Agani et al., 2000; Agani et al., 2002b; Chandel et al., 1998; Chandel et al., 2000; Sanjuan-Pla et al., 2005; Schroedl et al., 2002), myocyte contraction (Duranteau et al., 1998; Waypa et al., 2002), p38 activation (Kulisz et al., 2002), IL-6 production (Pearlstein et al., 2002), glutathione depletion (Mansfield et al., 2004), Na,K-ATPase activity (Dada et al., 2003), adipocyte differentiation (Carriere et al., 2004), and anoxia-regulated gene expression in yeast (Dirmeier et al., 2002). Furthermore, decreasing mtROS levels with mitochondrial inhibitors or ROS scavengers prevents these responses, indicating mtROS are necessary for hypoxic events. Increases in ROS generation during normoxia can also trigger hypoxic responses, including HIF-1α stabilization (BelAiba et al., 2004; Chandel et al., 2000; Goyal et al., 2004), suggesting that mtROS are sufficient to mimic hypoxia.

Despite this evidence, the mitochondria’s role in mammalian cellular O2 sensing has remained controversial, particularly because the use of pharmacological agents to affect hypoxic HIF-1α stabilization has produced conflicting data (Agani et al., 2002a; Agani et al., 2000; Chandel et al., 1998; Chandel et al., 2000) versus (Enomoto et al., 2002; Srinivas et al., 2001; Vaux et al., 2001). We investigated the controversy surrounding the use of pharmacological agents by extending previous observations. Our results with mitochondria-deficient rho zero (ρ°) cells and mitochondrial inhibitors support a role for mitochondria in cellular O2 sensing. Furthermore, using cytochrome c null embryonic cells, we clearly demonstrate that functional mitochondria are necessary to produce ROS and subsequently stabilize HIF-1α or HIF-2α in response to hypoxia. This novel genetic evidence indicates that mtROS act upstream of prolyl hydroxylases in regulating the stability of both HIF-1α and HIF-2α in mammalian cells.

Results

While mitochondria-deficient Hep3B and HEK293 ρ° cells were initially shown to be defective in hypoxic HIF-1α stabilization (Chandel et al., 1998; Chandel et al., 2000), later work suggested this may be an artifact due to selection with rotenone (Vaux et al., 2001) or mutation of nuclear genes. We chose to address this discrepancy by generating ρ° cells (King and Attardi, 1996), and avoiding selection with mitochondrial inhibitors. To minimize the impact of random mutation of nuclear genes, fresh pools of cells were generated for each experiment. We monitored ρ° status via mtDNA content by semi-quantitative PCR of the mitochondrially encoded genes, cytochrome oxidase subunit II (COXII), NADH dehydrogenase subunits I and II (ND1/2), and cytochrome b (CYTB), and Complex IV function via respirometry.

Hep3B cells required treatment with 100 ng/ml ethidium bromide (EtBr) for 3 weeks to disrupt mtDNA replication, resulting in loss of mtDNA (Figure ​(Figure1A1A and S1A) and inhibition of respiration (data not shown), compared to cells treated with 0 or 50 ng/ml EtBr. Upon exposure to hypoxia (1.5% O2), control cells treated with 0 or 50 ng/ml EtBr stabilized HIF-1α and HIF-2α while the ρ° cells exhibited an attenuated response. These cells retained HIF expression as treatment with the hypoxia mimetic desferroxamine (DFX) stabilized HIF-α similarly in all three populations of cells (Figure 1A). To ensure this was not a difference in cell type or severity of EtBr treatment, we also generated HEK293 ρ° cells similar to Chandel et al. (2000). Treatment of HEK293 cells with 50 ng/ml EtBr for 2 weeks depleted mtDNA and decreased cellular respiration by 95% (Figure ​(Figure1B1B and S1A). When subjected to hypoxia, control cells stabilized both HIF-α subunits while the ρ° cells failed to stabilize either HIF-α protein while responding equally well to DFX (Figure 1B). Similar results were obtained with HEK293T cells (Figure S1B).

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Functional mitochondria are required for hypoxic HIF-α stabilization.

A) Hep3B cells treated with 0, 50 or 100 ng/ml EtBr for 3 weeks and mtDNA levels determined via COXII PCR. Hep3B control (0 and 50) or ρ° (100) cells exposed to 4 hours of normoxia (21% O2), hypoxia (1.5% O2) or DFX (100 μM) and analyzed for HIF-1α and HIF-2α via western blot with γ-glutamylcysteine synthetase (GCS) as a loading control.

B) HEK293 cells treated for 2 weeks with 0 or 50 ng/ml EtBr analyzed for COXII mtDNA and respiration ((Resp, expressed as nmol O2 consumed/ml/min/106 cells). Control (0) or ρ° (50) cells exposed to 4 hours of normoxia, hypoxia or DFX.

C) 143B and 206ρ° cells analyzed for COXII mtDNA and respiration. Cells exposed to normoxia (N), hypoxia (H), hypoxia plus 100 ng/ml myxothiazol (M), or DFX (D) for 4 hours.

D, E) Hep3B cells treated with rotenone (D) or myxothiazol (E) and exposed to hypoxia or 100 μM CoCl2 for 4 hours. HIF-1α and HIF-2α levels determined and respiration measured to confirm mitochondrial inhibition.

It has been hypothesized that observed differences in ρ° hypoxic responses may arise through the use of freshly generated ρ° populations versus established cell lines that may have undergone metabolic adaptation or mutations in nuclear genes (Vaux et al., 2001). To address this concern, we obtained the human osteosarcoma-derived 143B parental cell line and 143B-206ρ° derivative used in previous studies (Srinivas et al., 2001; Vaux et al., 2001). ρ° status was confirmed by lack of mtDNA and respiration (Figure ​(Figure1C1C and S1A). In contrast to these previous reports which used more severe hypoxia (0.5-0.1% O2), 143B-206ρ° cells failed to stabilize HIF-α when exposed to 1.5% O2 for 4 hours, but responded to DFX. These findings indicate that mitochondrial electron transport is required for hypoxic HIF-α stabilization at 1.5%, supporting previous observations (Chandel et al., 1998; Chandel et al., 2000), and suggesting that differences in O2 tension may explain the discrepant results (see Discussion).

Due to concerns about long-term metabolic adaptation or genetic alteration of ρ° cells, we investigated the effect of acute mitochondrial inhibition by the Complex I inhibitor rotenone or the Complex III inhibitor myxothiazol, minimizing nonspecific effects by employing nanomolar doses. We expanded on prior experiments by using wider dose ranges, ensuring the treatments inhibited mitochondrial function for the duration of the experiment, and controlling for non-specific effects on HIF-α levels. Treatment of Hep3B cells with rotenone (Figure 1D) or myxothiazol (Figure 1E) for 4 hours inhibited respiration and prevented hypoxic HIF-1α and HIF-2α stabilization further supporting a role for mitochondria in this process. Of note, the highest doses of myxothiazol also affected HIF-α stabilization in response to CoCl2 (Figure 1E) highlighting the difficulties inherent to drug treatments that have prevented widespread acceptance of the mitochondrial O2 sensing model.

Therefore, we employed a genetic model to investigate the role of mitochondrial function in cellular O2 sensing, avoiding the nonspecific effects of pharmacological inhibition. Targeted mutagenesis of the murine somatic cytochrome c gene has previously been described (Li et al., 2000). Loss of cytochrome c prevents oxidation of cytochrome c1, keeping the Rieske Iron-sulfur protein reduced. This prevents oxidation of ubiquinol and formation of the ubisemiquinone radical, thus eliminating one important source of superoxide implicated in hypoxic HIF-1α stabilization (Chandel et al., 1998; Chandel et al., 2000). Although this mutation results in embryonic death by day 10.5, we generated wildtype (WT), heterozygous (Het), and null cell lines from a representative litter of day 8.5 embryos (Figure 2A). WT and Het lines exhibited similar respiratory rates, while null cells were devoid of any measurable mitochondrial O2 consumption (Figure 2C), or cytochrome c protein (Figure 2B).

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Cytochrome c null embryonic cells are deficient in their hypoxic response.

A) Genotype of individual cell lines determined via PCR.

B) Cytochrome c protein expression determined via western blot.

C) Mitochondrial respiration was measured.

D) Embryonic cells mounted in a flow-through chamber and ROS production in response to 1% O2 was monitored with DCFH-DA. mtROS levels determined by treatment with 100 ng/ml myxothiazol.

E) Embryonic cells exposed to normoxia (N), hypoxia (Hyp, 1.5% O2) or 100 μM DFX for 4 hours in the presence or absence of 100 ng/ml myxothiazol and HIF-α levels determined. Densitometry analysis of HIF-1α levels in 5 separate experiments normalized to normoxic controls.

We first investigated real-time, in vivo ROS production in these cell lines through the use of the oxidant-sensitive dye dichlorodihydrofluorescein (DCFH; Figure 2D), and an ROS-sensitive FRET probe (HSP-FRET; data not shown) utilizing single cell epifluorescence microscopy. Whereas both cell lines exhibited similar basal levels of ROS production, WT cells dramatically increased DCFH oxidation upon exposure to hypoxia while cytochrome c null cells exhibited an attenuated response (Figure 2D). Importantly, treatment of the null cells with Complex III inhibitors myxothiazol or stigmatellin elicited no change in ROS production, while WT cells treated with inhibitors decreased their hypoxic ROS production to that observed for null cells (Figure 2D, and data not shown). Furthermore, null cells were unable to stabilize HIF-1α or HIF-2α in response to hypoxia in direct contrast to the WT and Het cells (Figure 2E) but retained response to DFX, with HIF α subunits appearing as multiple bands due to post-translational modifications. Myxothiazol (Figure 2E) or rotenone (Figure S1C) abolished hypoxic but not DFX-induced HIF-α stabilization in cytochrome c expressing cells. This argues strongly that the increased ROS produced in the hypoxic WT cells are mitochondrial, and supports the hypothesis that mtROS generated by Complex III are responsible for hypoxic HIF-α stabilization.

Stable reintroduction of cytochrome c into an independent null cell line restored respiration (Figure 3A) and hypoxic mtROS accumulation (Figure 3B) as measured by an in vitro end-point assay. This correlated with a restored ability to properly stabilize HIF-1α to levels similar to WT, in a mitochondria-dependent manner, and in multiple clones (Figure 3C). These results suggest that the inability of null cells to properly sense O2 is due to mitochondrial dysfunction resulting from cytochrome c loss.

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Reintroduction of cytochrome c restores hypoxic response.

A) An independent cytochrome c null cell line stably transfected with a control (Hyg), or cytochrome c expression vector (CytC) and assayed for cytochrome c protein levels and respiratory activity.

B) ROS accumulation monitored with carboxy-H2 DCFDA and fluorescence determined (+/− S.E) after 4 hours of hypoxia.

C) Cells exposed to 4 hours of normoxia (N), 100 μM DFX (D), or hypoxia in the absence (H) or presence (M) of 100 ng/ml myxothiazol and HIF-1α expression determined.

All embryonic cell lines responded similarly to DFX suggesting that cytochrome c null cells maintain normal HIF-α transcription, translation, and degradation. However, to test if they continue to hydroxylate and degrade HIF-α under hypoxic conditions, cytochrome c WT or null cells were treated with 1.5% O2 in the presence of the proteosome inhibitor MG132. While the null cells failed to stabilize HIF-α in response to hypoxia, inhibition of proteosome degradation by MG132 led to HIF-α accumulation similar to WT (Figure 4A). This demonstrates that hydroxylation and degradation of HIF-α continued to occur in the cytochrome c null cells even at this reduced O2 concentration.

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Proteosome inhibition, anoxia, or H2O2 is sufficient to stabilize HIF-α in cytochrome c null cells.

A) Cytochrome c WT or null cells exposed to 4 hours of normoxia or hypoxia in the presence of 10 μM MG132 or 100 μM DFX and HIF-α levels determined.

B) Embryonic cells exposed to 21%, 1.5%, and 0% O2, or 100 μM DFX for 3 hours.

C) Hep3B cells treated with H2O2 or exposed to hypoxia (H) for 2 hours.

D) Hep3B cells treated with glucose oxidase enzyme (Gluc Ox) or CoCl2 for 2 hours in the presence or absence of 100 units/ml catalase.

E) Cytochrome c null cells treated with boluses of t-Butyl hydroperoxide (TBP) every 15 mins or a single dose of CoCl2 (100 μM) for 1 or 2 hours.

The mitochondrial dependence of HIF-α stabilization can also be bypassed by severe O2 deprivation (anoxia ≈ 0% O2) where prolyl hydroxylases appear to be substrate limited (Schroedl et al., 2002). While the null cells failed to stabilize HIF-1α or HIF-2α in response to 1.5% O2, exposure to 0% O2 induced both HIF-α subunits in a mitochondria-independent manner (Figure 4B), suggesting functional mitochondria are not necessary for cellular responses to severe O2 deprivation in which the hydroxylases are directly inhibited.

To test whether ROS production is also sufficient to stabilize HIF-1α, Hep3B cells were treated with exogenous hydrogen peroxide (H2O2) as previously described (Chandel et al., 2000). Doses as low as 25 μM stabilized HIF-1α as well or better than hypoxia (Figure 4C). In addition, incubation of Hep3B cells with the H2O2-generating enzyme, glucose oxidase, was also sufficient to stabilize HIF-1α and HIF-2α. This was an H2O2-dependent event as exogenous catalase abolished HIF-α stabilization (Figure 4D). To ensure the effects of exogenous H2O2 were not mediated by mitochondria, cytochrome c null cells were treated with t-Butyl hydroperoxide (TBP), a stable H2O2 analogue. Even in the absence of functional mitochondria, exogenous H2O2 induced HIF-1α and HIF-2α stabilization similar to the hypoxia mimetic CoCl2 (Figure 4E). These results indicate that ROS production is sufficient for HIF-α stabilization and functions downstream of the mitochondria.

Discussion

Cytochrome c null cells provide novel genetic evidence supporting a role for mtROS in the mammalian cellular O2 sensing pathway, without the use of EtBr or pharmacological inhibitors. Utilizing this genetic model of mitochondrial dysfunction, we show that ROS production is necessary and sufficient to promote HIF-α stabilization, presumably acting through the prolyl hydroxylases. While the null cells appear to produce some hypoxic ROS, it is insufficient to stabilize HIF-α, suggesting that either the cellular compartment(s) participating in redox signaling, the type, or the magnitude of the ROS signal, is important. It should also be noted that in contrast to studies in yeast (Barros et al., 2003), and isolated mitochondria (Zhao and Xu, 2004), loss of cytochrome c did not lead to increased basal levels of ROS production. Whether this is due to species differences or adaptation, the salient result is that null cells fail to sufficiently increase mtROS production upon hypoxic exposure, correlating with an inability to properly stabilize HIF-α. While ROS are involved in the hypoxic response and can induce HIF-α in a mitochondria-independent manner, other mitochondria-derived factors also clearly play a role in modulating hydroxylase activity (Selak et al., 2005).

It was previously shown that HIF-1α stabilization is mitochondria-independent during severe O2 limitation (Schroedl et al., 2002), and we have confirmed those results with cytochrome c null cells. This observation may explain some of the apparent discrepancies in the literature regarding the use of ρ° cells and pharmacological inhibitors. Indeed, we have clearly shown (using ρ° cells, mitochondrial inhibitors, and cytochrome c null cells) that at 1.5% O2 the mitochondria play a vital role in proper cellular O2 sensing, supporting previous observations (Agani et al., 2000; Agani et al., 2002b; Chandel et al., 1998; Chandel et al., 2000; Schroedl et al., 2002). The discrepancies arose when comparing these results to those obtained under more severe hypoxic stress (Enomoto et al., 2002; Srinivas et al., 2001; Vaux et al., 2001) in which it appears the hydroxylases are directly responsible for O2 sensing and HIF-α stabilization. These results emphasize the need to accurately define the levels of O2 and consider the effects of various O2 tensions when investigating hypoxic phenomena.

It is possible that increased O2 availability in the respiratory-deficient cytochrome c null cells accounts for differences in sensitivity to hypoxia, similar to that recently reported with nitric oxide-mediated (NO) mitochondrial inhibition (Hagen et al., 2003). However, we have found that modulation of mitochondrial O2 consumption with the Complex IV inhibitor KCN, or the mitochondrial uncoupler FCCP is insufficient to impact HIF-1α stabilization in Hep3B or HEK293 cells (Figure S2). Furthermore, while intracellular O2 gradients have been shown to exist near mitochondrial clusters, other organelles and the cytoplasm (where prolyl hydroxylases are reported to reside; (Metzen et al., 2003) have O2 levels similar to the surrounding media which do not change upon mitochondrial inhibition (Jones, 1986; Jones and Mason, 1978). Of note, a mitochondrially-targeted antioxidant has recently been shown to block hypoxic mtROS production and HIF-1α stabilization without affecting cellular respiration (Sanjuan-Pla et al., 2005). Taken together with our results, this suggests that upon mitochondrial inhibition it is the inability to produce mtROS from Complex III during hypoxia that results in the O2 sensing defect. Furthermore, we demonstrate that exogenous ROS can lead to the stabilization of HIF-α even when O2 levels are high, providing further proof that mtROS are important components of cellular O2 sensing pathways.

How mtROS affect hydroxylase activity is critical to understanding cellular O2 sensing. ROS are known to be involved in a number of signaling pathways (Thannickal and Fanburg, 2000) that potentially modulate hydroxylase activity. Alternatively, ROS production could result in redox-mediated events within the cell. Recently, redox changes through the depletion of ascorbate by CoCl2 (Salnikow et al., 2004), or via oxidation of cellular iron by persistent ROS production (Gerald et al., 2004), have been shown to inhibit hydroxylase activity leading to stabilization of HIF-α subunits.

Our genetic model in which the hypoxic response has been altered such that cells retain functional prolyl hydroxylases but lack the ability to properly sense and signal O2 deprivation provides a unique tool to investigate the in vivo regulation of the prolyl hydroxylases. Indeed, our work suggests that in vitro models (Hirsila et al., 2003; Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001) may fail to fully recapitulate in vivo cellular O2 sensing, since in the absence of functional mitochondria, hydroxylases appear to efficiently target HIF for degradation at 1.5% O2 intracellularly. It is likely that an array of mammalian cellular O2 sensors exist, controlling various hypoxic responses depending on the cell type, O2 tension, and other cellular conditions. Through the use of cytochrome c null cells, we have confirmed that mitochondria are essential for hypoxic HIF-1α stabilization at 1.5% O2 and show the same is true for HIF-2α. This, along with other emerging genetic models (Brunelle et al., 2005; Guzy et al., 2005), will provide valuable tools for investigating the role of the mitochondria in other hypoxic phenomena.

Experimental Procedures

Tissue Culture Cells and Reagents

Hep3B, HEK293, and 143B cell lines were maintained in Dulbecco’s Modified Eagles Medium (DMEM; CellGro), 10% fetal bovine serum (FBS; GemCell), 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM MEM non-essential amino acids, and 1mM sodium pyruvate. Murine embryonic cells were maintained in DMEM with 4.5 grams glucose/ml, 20% FBS (Hyclone), 25 mM HEPES, 2 mM glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.1 mM MEM non-essential amino acids, 50 μM beta-mercaptoethanol, 500-1000 U/ml mouse LIF, 2 mM sodium pyruvate, and 50 μg/ml uridine.

Hypoxia and Anoxia

Hypoxia (1.5% O2, 5% CO2, 93.5% N2) was achieved using an In Vivo2 hypoxic workstation (Ruskinn Technologies, Leeds, UK), with all extracts prepared inside the workstation to prevent reoxygenation. Anoxic experiments were carried out in Coy Laboratories glove-box workstations equilibrated to either hypoxia (1.5% O2, 5% CO2, 93.5% N2) or anoxia (5% CO2, 3-4% H2, balance N2), with a palladium catalyst.

Rho Zero (ρ°) Generation

Cultures were treated with ethidium bromide (EtBr; 50 or 100 ng/ml) in complete DMEM supplemented with 0.1 mM MEM non essential amino acids (Gibco), 2.5 mM sodium pyruvate, and 50 μg/ml uridine (King and Attardi, 1996). Cells were considered ρ° when there was no detectable mtDNA by PCR and no mitochondria-dependent O2 consumption. O2 consumption rate was measured in a water-jacketed respirometer chamber by using an oxygen electrode (Hansatech Instruments, Norfolk England) at 37°C, subtracting out KCN-resistance O2 consumption to calculate respiratory activity.

Western Blots

Whole cell lysates were prepared in 50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, and complete protease inhibitor (Roche Molecular Biochemicals). Antibodies used were: α-Human HIF-1α (Transduction Laboratories), α-murine HIF-1α (Simon Laboratory), α-HIF-2α (Novus), α-cytochrome c (BD PharMingen), and α-GCS heavy subunit (Neomarkers).

Mouse Embryonic Cells

Murine embryonic cells were generated as previously described (Li et al., 2000). Briefly, day 8.5 embryos were dissected free of maternal tissues, minced, and treated with 0.25% trypsin with EDTA (Gibco) for 5-15 mins at 37°C. After trypsin inactivation with FBS, the cells were plated onto mitomycin-C treated mouse embryonic fibroblasts (MEFs) and after 2-3 weeks, adapted for growth on gelatin. Genotyping was performed as previously described (Li et al., 2000). Cytochrome c expression was restored to the null cells by electroporation with a full-length murine cDNA encoding the somatic cytochrome c under the control of the EF-1α promoter, and selection of stable clones with hygromycin, with the empty vector serving as a control.

ROS Measurements

For real-time ROS measurements, cells on glass coverslips were mounted in a flow-through chamber and perfused with 5 μM DCFH-DA in a basic salt solution bubbled with a 21% O2, 5% CO2, 74% N2 gas mixture. Hypoxia was induced by bubbling with a 1% O2, 5% CO2, 96% N2 gas mixture. Fluorescence was monitored with an inverted epifluorescence microscope. For the end-point assay, cells were incubated in PBS with 20 μM 5-(and-6)-carboxy-2′, 7′- dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). Cells were washed from the plate in the incubation medium, and transferred to an opaque 96 well plate, excited at 488 nm, and fluorescence emission measured at 530 nm using a Fluoromax-2 fluorimeter (Jobin Yvon Spex, Edison, NJ). Hypoxic samples were harvested under hypoxic conditions in the Ruskinn In Vivo2 workstation, and the plate kept sealed prior to reading.

Supplementary Material

Supplementary Data 1

Supplementary Data 2

Supplementary Figure 1

Supplementary Figure 2

Acknowledgements

We thank Brian Keith for critical review of the manuscript and helpful discussions. 143B and 143B-206ρ° cells were generously provided by M.P. King. Funding was provided by the American Heart Association (KDM), Abramson Family Cancer Research Institute (KDM, YP, RMY, MCS), National Institutes of Health (MCS, PTS), and Howard Hughes Medical Institute (YP, RMY, MCS).

Footnotes

Supplemental Data Supplemental Data including Supplemental Experimental Procedures and two Figures and can be found with this article online at http://www.cellmetabolism.org/

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