Supplementary Materials

Supplementary Material for:

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models

Saravanan S. Karuppagounder, Ishraq Alim, Soah J. Khim, Megan W. Bourassa, Sama F. Sleiman, Roseleen John, Cyrille C. Thinnes, Tzu-Lan Yeh, Marina Demetriades, Sandra Neitemeier, Dana Cruz, Irina Gazaryan, David W. Killilea, Lewis Morgenstern, Guohua Xi, Richard F. Keep, Timothy Schallert, Ryan V. Tappero, Jian Zhong, Sunghee Cho, Frederick R. Maxfield, Theodore R. Holman, Carsten Culmsee, Guo-Hua Fong, Yijing Su, Guo-li Ming, Hongjun Song, John W. Cave, Christopher J. Schofield, Frederick Colbourne, Giovanni Coppola, Rajiv R. Ratan*

*Corresponding author. E-mail: rrr2001{at}med.cornell.edu

Published 2 March 2016, Sci. Transl. Med. 8, 328ra29 (2016)
DOI: 10.1126/scitranslmed.aac6008

This PDF file includes:

  • Materials and Methods
  • Fig. S1. Hemin, the reactive ferric protoporphyrin IX group of hemoglobin, induces concentration-dependent death in multiple cell types.
  • Fig. S2. CPO or a CPO analog (5342) differentially inhibit HIF-PHD activity, although they have a similar affinity for iron.
  • Fig. S3. Conditional reduction of HIF-PHD1, HIF-PHD2, and HIF-PHD3 in the striatum does not affect ICH-induced hematoma size or brain edema.
  • Fig. S4. Canonical HIF-PHD inhibitors DFO, CPO, and DHB do not stabilize ODD-luciferase in brains of mice.
  • Fig. S5. Adaptaquin binds to and inhibits recombinant PHD2.
  • Fig. S6. Adaptaquin does not inhibit collagenase activity in vivo.
  • Fig. S7. Adaptaquin significantly reduced ICH-induced edema.
  • Fig. S8. Adaptaquin’s beneficial effects on ICH outcomes are not associated with changes in core body temperature.
  • Fig. S9. Reduction of HIF-1 α or HIF-2 α by RNA interference does not potentiate hemin-induced toxicity or affect the ability of chemically diverse HIF-PHD inhibitors to prevent hemin toxicity.
  • Fig. S10. Hemin-induced neuronal death is abrogated by the HIF-PHD inhibitor adaptaquin.
  • Fig. S11. Adaptaquin inhibits glutamate-induced mitochondrial dysfunction.
  • Fig. S12. Adaptaquin is distinct from the classical antioxidant N-acetylcysteine in abrogating ATF4-mediated death.
  • Fig. S13. Adaptaquin does not affect global histone acetylation or methylation or inhibit 12-lipoxygenase activities.
  • Fig. S14. Adaptaquin does not affect 3-nitrotyrosine, a biomarker of oxidative stress.
  • Fig. S15. Adaptaquin does not affect TET enzyme activity, a subfamily of the iron-, 2-oxoglutarate–, and oxygen-dependent dioxygenases that demethylate DNA, at concentrations where it completely abrogates neuronal death.
  • Fig. S16. Adaptaquin protection is not associated with induction of HIF-dependent genes VEGF and EPO in the striatum of mice.
  • Table S1. Inability of adaptaquin to influence likely off-target enzymes at concentrations where it fully protects neurons.
  • References (51, 52)

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