Volume 1, Issue 6, Pages 393-399 (June 2005) Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-α activation  Kyle D. Mansfield, Robert D. Guzy, Yi Pan, Regina M. Young, Timothy P. Cash, Paul T. Schumacker, M. Celeste Simon  Cell Metabolism  Volume 1, Issue 6, Pages 393-399 (June 2005) DOI: 10.1016/j.cmet.2005.05.003 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 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 ρ0 (100) cells exposed to 4 hr 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 ρ0 (50) cells exposed to 4 hr of normoxia, hypoxia, or DFX. C) 143B and 206ρ0 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 hr. D and E) Hep3B cells treated with rotenone (D) or myxothiazol (E) and exposed to hypoxia or 100 μM CoCl2 for 4 hr. HIF-1α and HIF-2α levels determined and respiration measured to confirm mitochondrial inhibition. Cell Metabolism 2005 1, 393-399DOI: (10.1016/j.cmet.2005.05.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 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 (± SEM). 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 hr in the presence or absence of 100 ng/ml myxothiazol and HIF-α levels determined. Densitometry analysis of HIF-1α levels in five separate experiments normalized to normoxic controls (± SEM). Cell Metabolism 2005 1, 393-399DOI: (10.1016/j.cmet.2005.05.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 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 (± SEM) after 4 hr of hypoxia. C) Cells exposed to 4 hr 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. Cell Metabolism 2005 1, 393-399DOI: (10.1016/j.cmet.2005.05.003) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 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 hr of normoxia or hypoxia in the presence of 10 μM MG-132 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 hr. C) Hep3B cells treated with H2O2 or exposed to hypoxia (H) for 2 hr. D) Hep3B cells treated with glucose oxidase enzyme (Gluc Ox) or CoCl2 for 2 hr 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 hr. Cell Metabolism 2005 1, 393-399DOI: (10.1016/j.cmet.2005.05.003) Copyright © 2005 Elsevier Inc. Terms and Conditions