1. Oxygen-dependent ATF-4 stability is mediated by the PHD3 oxygen sensor
by Jens Köditz, Jutta Nesper, Marieke Wottawa, Daniel P. Stiehl, Gieri Camenisch, Corinna Franke, Johanna Myllyharju, Roland H. Wenger, and Dörthe M. Katschinski
Blood
Volume 110(10):
November 15, 2007
©2007 by American Society of Hematology

2. Interaction of ATF-4 with PHD3.
Interaction of ATF-4 with PHD3. (A) The yeast reporter strain MaV203 expressing Gal4-BD-PHD3 and Gal4-AD-ATF-4 aa 27–351 or Gal4-AD-HIF2α aa 73–870 was assayed for histidine auxotrophy. (B) His-PHD3 expressed in insect cells is captured by immobilized MBP–ATF-4. Antibodies against MBP or PHD3 were used for immunoblot detection as indicated. (C) HeLa cells were cotransfected with a firefly luciferase reporter gene plasmid driven by HREs together with a constitutively expressed renilla luciferase control vector as depicted and the expression plasmids pcDNA3.1HIF-2α, pcDNA3.1PHD3, or pcDNA3.1V5-ATF-4. After 24 hours, HeLa cells were lysed and analyzed for firefly (FL) and renilla luciferase (RL) activities. (D) HeLa cells were cotransfected with an HRE-driven firefly luciferase reporter gene plasmid and a constitutively expressed renilla luciferase control vector as in panel C and the empty control vector pcDNA3.1 or the expression plasmid pcDNA3.1V5ATF-4. Subsequently, cells were incubated at 20% O2 or 1% O2 for 24 hours, lysed, and analyzed for firefly and renilla luciferase activities (C,D). Shown are mean values of the FL/RL ratios (± SD) of 3 independent experiments.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

3. Mapping the PHD3 binding domain of ATF-4.
Mapping the PHD3 binding domain of ATF-4. (A) Schematic drawing of the protein domains of ATF-4. Arrows (27, 42, 51, and 83) indicate the insertion of the 4 independent ATF-4 prey-clones identified by yeast 2-hybrid screening of the human brain cDNA library with PHD3 as bait. (B) Yeast reporter strain MaV203 expressing Gal4-BD-PHD3 and various domains of ATF-4 as Gal4-AD-fusions was assayed for histidine and uracil auxotrophy. (C) Yeast reporter strain MaV203 expressing Gal4-BD-PHD3 and various deletion variants of ATF-4 as Gal4-AD fusions was assayed for histidine and uracil auxotrophy.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

4. Hypoxia as well as proteasome and PHD inhibition stabilize ATF-4.
Hypoxia as well as proteasome and PHD inhibition stabilize ATF-4. (A) HeLa cells were treated for 8 hours with 300 nM thapsigargin or 10 μM MG132. Subsequently, cells were lysed and analyzed by immunoblotting. (B) HeLa cells were transiently transfected with an ATF-dependent firefly (FL) luciferase reporter gene plasmid (pATFx2-Luc) together with the renilla luciferase (RL) control plasmid pRLSV40. Following exposure to 20% or 1% O2, with or without the addition of 1 mM DMOG, cells were lysed and luciferase activities were determined. (C) TS-20 cells (with a temperature-sensitive E1 ubiquitin–activating enzyme defect) and E1-reconstituted H38–5 cells were exposed to 34°C or 39°C. Subsequently, cells were lysed and analyzed by immunoblotting. Shown are mean values of the FL/RL ratios (± SD) of 3 independent experiments.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

5. Kinetics of hypoxic stabilization and reoxygenation-induced destabilization of ATF-4.
Kinetics of hypoxic stabilization and reoxygenation-induced destabilization of ATF-4. (A) HeLa cells were incubated at 20%, 1%, or 0.2% O2, with or without the addition of 1 mM DMOG. After 4 or 24 hours, the cells were lysed and analyzed by immunoblotting. As a control for the induction of ATF-4 by ER stress, one dish of HeLa cells was treated with 300 nM thapsigargin for 4 hours. (B) HeLa cells were incubated at 0.2% O2 with or without the addition of 1 mM DMOG. At 4 hours later, 20 μg/mL cycloheximide was added, and cells were reoxygenated at 20% O2. After the indicated time points, cells were lysed and analyzed by immunoblotting.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

6. ATF-4 aa 154–181 harbors a novel ODD domain.
ATF-4 aa 154–181 harbors a novel ODD domain. (A) HeLa cells were transiently transfected with V5–ATF-4, V5–ATF-3, or V5–ARNT expression vectors. To control for equal transfection efficiencies, cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 1% O2 or treated with 1 mM DMOG for 24 hours. Subsequently, cells were lysed and analyzed by immunoblotting. (B) HeLa cells were transiently transfected with different ATF-4 variants and treated as in panel A. (C) HeLa cells were transiently transfected with V5–ATF-4 or V5–ATF-4Δ aa To control for equal transfection efficiencies, cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 1% O2. After exposure to 1% O2 for 24 hours, the cells were treated with 20 μg/mL cycloheximide and were reoxygenated with 20% O2. Cells were lysed at different reoxygenation time points, and total protein was analyzed by immunoblotting. Band intensities of 3 independent experiments were determined and half-life of the V5–ATF-4 and V5–ATF-4Δ aa proteins were calculated. (D) Decline of V5–ATF-4 and V5–ATF-4Δ aa after reoxygenation calculated from 3 independently performed experiments as described in panel C. Shown are mean values (± SD) of 3 independent experiments.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

7. The oxygen-dependent stability of ATF-4 is proline dependent.
The oxygen-dependent stability of ATF-4 is proline dependent. (A) HeLa cells were transiently transfected with a constitutively expressed renilla luciferase (RL) control vector, an expression plasmid containing the Gal4 AD/BD (pM3VP16) fused to ATF-4 aa 133–183; ATF-4 aa , in which prolines P156, P162, P164, P167, and P174 have been mutated to alanine; HIF-2α aa ; or HIF-2α aa 404–568 P405/531A together with the Gal4-driven firefly luciferase (FL) plasmid pGRExE1bLuc. After transfection cells, were exposed to 20% or 1% O2 for 24 hours, lysed, and analyzed for FL and RL activities. Shown are mean values plus or minus SD of the FL/RL ratios of 3 independent experiments. (B) HeLa cells were transiently transfected with V5–ATF-4, V5–ATF-4Δ aa , or V5–ATF-4 5 × P > A, in which prolines P156, P162, P164, P167, and P174 have been mutated to alanine. To control for equal transfection efficiencies cells were cotransfected with pEGFPC1. At 6 hours after transfection, cells were exposed to 20% or 1% O2 for 24 hours. Subsequently, the cells were lysed and analyzed by immunoblotting. (C) Multiple sequence alignment of the proposed ATF-4 ODD domain.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology

8. ATF-4 expression is controlled by PHD3.
ATF-4 expression is controlled by PHD3. (A) HeLa cells were transiently transfected with siRNA targeting PHD2 or PHD3 and exposed to 20% or 1% O2. After 24 hours, the cells were lysed and analyzed by immunoblotting. One dish of HeLa cells was treated for 8 hours with 300 nM thapsigargin (t). Subsequently, cells were lysed and analyzed together with the protein extracts of the siRNA-transfected cells. (B) Hela cells were transiently transfected with siRNA targeting PHD3 or a control siRNA and exposed to 1% O2 for 4 hours with subsequent reoxygenation. After the indicated time points, cells were lysed and analyzed by immunoblotting. (C) HeLa cells were transiently transfected with siRNA targeting PHD2 or PHD3 and exposed to 20% or 1% O2. After 24 hours, the cells were lysed and total RNA was extracted and analyzed for ATF-4, PHD2, and PHD3 mRNA levels by real-time reverse transcriptase (RT)–PCR. Shown are mean values plus or minus SD of 3 independent experiments of ratios to the ribosomal protein L28 mRNA levels, which were used as housekeeping controls.
Jens Köditz et al. Blood 2007;110:
©2007 by American Society of Hematology