1. 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 (June 2005)
DOI: /j.cmet
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. 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, DOI: ( /j.cmet )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. 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, DOI: ( /j.cmet )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. 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, DOI: ( /j.cmet )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. 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, DOI: ( /j.cmet )
Copyright © 2005 Elsevier Inc. Terms and Conditions