1. Silencing of eIF3e promotes blood perfusion recovery after limb ischemia through stabilization of hypoxia-inducible factor 2α activity 
Takuya Hashimoto, MD, PhD, Li Chen, PhD, Hideo Kimura, MD, PhD, Alexander Endler, PhD, Hiroyuki Koyama, MD, PhD, Tetsuro Miyata, MD, PhD, Futoshi Shibasaki, MD, PhD, Toshiaki Watanabe, MD, PhD 
Journal of Vascular Surgery 
Volume 64, Issue 1, Pages e3 (July 2016)
DOI: /j.jvs
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2. Fig 1
Effects of eukaryotic translation initiation factor 3 subunit e (eIF3e) silencing on murine primary skeletal muscle myoblasts (MSMMs), assessed by immunoblotting. a, Whole-cell lysates were prepared from MSMMs at 24 hours after transfection with the eIF3e-silencing plasmid (right) or control plasmid (left) to investigate endogenous eIF3e and endogenous hypoxia-inducible factor 2α (HIF-2α) protein expression. b, As supportive data, whole-cell lysates were prepared from MSMMs at 48 hours after cotransfection with pcDNA3-Myc-HIF-2α and the eIF3e-silencing plasmid (right) or control plasmid (left) to investigate endogenous eIF3e and exogenous HIF-2α protein expression.
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3. Fig 2
Messenger RNA (mRNA) expression of (a) basic fibroblast growth factor (bFGF), (b) platelet-derived growth factor B (PDGF-B), (c) vascular endothelial growth factor A (VEGF-A), (d) hepatocyte growth factor (HGF), and (e) monocyte chemoattractant protein 1 (MCP-1) in murine primary skeletal muscle myoblasts (MSMMs) at 18 hours and 42 hours after treatment with random-sequence small interfering RNA (siRNA; left column) and with eukaryotic translation initiation factor 3 subunit e (eIF3e) siRNA (right column). *P < .05 vs random siRNA-treated group (n = 3).
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4. Fig 3
Assessment of paracrine activity of eukaryotic translation initiation factor 3 subunit e (eIF3e)-silenced human primary skeletal muscle myoblasts (HSMMs). a, Human umbilical vein endothelial cell (HUVEC) tube formation assay. Top photographs, HUVECs cultured in 24-hour conditioned medium of eIF3e-silenced HSMMs. Bottom photographs, HUVECs cultured in 24-hour conditioned medium of HSMMs transfected with control plasmid (×20; n = 5). Bar indicates 100 μm. b-d, Secreted proteins were measured from the supernatant of HSMMs 24 hours after transfection. Protein levels were measured with MUSTag kits (n = 3). *P < .05, **P < .01 vs random-sequence small interfering RNA (siRNA)-treated group. Compared with controls, the level of basic fibroblast growth factor (bFGF) increased from 393 to 676 pg/mL (P = .046), whereas the level of angiopoietin 1 (ANG-1) increased from 251 to 281 pg/mL (P = .049) after eIF3e silencing. Vascular endothelial growth factor A (VEGF-A) level did not show significant changes.
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5. Fig 4
Effects of eukaryotic translation initiation factor 3 subunit e (eIF3e) silencing on clinical recovery of ischemic limbs. *P < .05 vs random-sequence small interfering RNA (siRNA)-treated group (n = 9). a, Clinical scoring of ambulatory impairment. b, Clinical scoring of tissue damage.
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6. Fig 5
Effect of eukaryotic translation initiation factor 3 subunit e (eIF3e) silencing on perfusion recovery of ischemic limbs. a, Representative photographs of limb perfusion recovery. b, Ratio of ischemic (left) to nonischemic (right) paw blood flow. *P < .05 vs random-sequence small interfering RNA (siRNA)-treated group (n = 9).
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7. Supplementary Fig (online only)
Schema of hypoxia-inducible factor 2α (HIF-2α) protein activity and degradation. Under normoxic conditions, the HIF-α subunits are rapidly degraded by the proteasome system after its ubiquitination by the von Hippel-Lindau protein (pVHL) that acts as its E3 ligase. The interaction of HIF with pVHL is greatly enhanced by the hydroxylation of prolyl residues located in the oxygen-dependent degradation domain of the proteins. In the HIF-2α isoform, Pro-405 and Pro-531 residues are the substrates for hydroxylation that are mediated by specific prolyl hydroxylase enzymes acting as oxygen sensors. Under conditions of oxygen sufficiency, the HIF-α proteins are hydroxylated, ubiquitinated, and degraded by the proteasome system. During hypoxia, HIF-α accumulates, dimerizes with β subunit, and forms the transcriptionally active HIF-α complex. On the other hand, eukaryotic translation initiation factor 3 subunit e (eIF3e) plays a subtype-specific role in post-transcriptional regulation of HIF-2α. Binding of eIF3e to IBS (Int6/eIF3e binding site) leads to proteasomal degradation of HIF-2α protein irrespective of hypoxia or normoxia. Thus, inhibition of eIF3e by small interfering RNA (siRNA) leads to HIF-2α protein accumulation. bHLH, Basic helix-loop-helix; CTAD, C-terminal transcription activation domain; FIH, factor-inhibiting HIF; HRE, hypoxia response element; NTAD, N-terminal transcription activation domain; PAC, PAS-associated C-terminal domain; PAS, Per/Ahr-ARNT/Sim; PHD1-3, prolyl hydroxylases.
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8. Journal of Vascular Surgery 2016 64, 219-226. e3DOI: (10. 1016/j. jvs
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