1. Volume 38, Issue 6, Pages 864-878 (June 2010)
Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α 
Ji-Hong Lim, Yoon-Mi Lee, Yang-Sook Chun, Junjie Chen, Ja-Eun Kim, Jong-Wan Park 
Molecular Cell 
Volume 38, Issue 6, Pages (June 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 38, 864-878DOI: (10.1016/j.molcel.2010.05.023)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 HIF-1α Interacts with SIRT1
(A) HEK293T cells were transfected with plasmids indicated, and cell extracts were immunoprecipitated with anti-Myc, and coprecipitated HA-HIF-1α was analyzed using anti-HA.
(B) After HT1080 and HEK293 were incubated under normoxic or hypoxic conditions for 8 hr, coprecipitation of endogenous SIRT1 and HIF-1α was performed using anti-SIRT1 and anti-HIF-1α.
(C) SIRT1 and Flag-HIF-1α fragments (amino acids; NT1, 1–200; NT2, 201–400; ODDD, 401–600; CT, 601–826) expressed in HEK293T were immunoprecipitated.
(D) Recombinant GST-HIF-1α constructs were incubated with recombinant His-SIRT1 in vitro and pulled down using Ni2+-affinity beads.
(E) Structures of SBP/Flag-tagged SIRT1 constructs are schematically shown. Black box represents catalytic domain.
(F) HEK293T cells were cotransfected with plasmids of SBP/Flag-SIRT1s and HA-HIF-1α N terminus (NT, aa 1–400) or C terminus (CT, aa 577–826). Lysates were immunoprecipitated with anti-HA.
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4. Figure 2 HIF-1α Is Acetylated by PCAF and Deacetylated by SIRT1
(A and B) After transfected with SIRT1 siRNAs (A) or plasmid (1–3 μg) (B), HT1080 cells were incubated under normoxic or hypoxic conditions for 8 hr. Endogenous HIF-1α was immunopreciptated with anti-HIF-1α, and acetylated HIF-1α was identified using anti-acetyl-lysine.
(C) HEK293T cells were transfected with HA-tagged HIF-1α or K532R, and incubated with 10 μM MG132 and/or 5 mM NAM for 8 hr. Acetylated HIF-1α was immunoprecipitated with anti-HA.
(D and E) HT1080 cells, which had been transfected with PCAF siRNAs (D) or plasmid (E), were incubated in normoxia or hypoxia for 8 hr, and acetylated HIF-1α was identified by immunoprecipitation and immunoblotting.
(F) HT1080 cells, which had been transfected with 1 μg of Flag-PCAF and/or 1 to 3 μg of Myc/His-SIRT1, were incubated 8 hr, and acetylated HIF-1α was detected.
(G) HEK293T cells were cotransfected with HA-HIF-1α and Flag-PCAF, and acetylated (Ac) HIF-1α was purified by HA-affinity chromatography and elution with HA peptide. Unacetylated (UnAc) HIF-1α was produced without Flag-PCAF. HA-HIF-1αs were incubated with His-SIRT1 in the presence of NAD+ (0.1 or 0.5 mM) or NAM (5 mM).
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5. Figure 3 Acetylation of Lys674 Is Regulated by PCAF and SIRT1
(A) HEK293T cells were transfected with a Flag-HIF-1α peptide and subjected to 8 hr hypoxia. Flag-HIF-1α peptides were purified by Flag-affinity chromatography and elution with Flag peptide, and its lysyl acetylation was identified using anti-acetyl-lysine.
(B) Acetylated (Ac-) or unacetylated (UnAc-) HIF-1α peptides were produced, purified, in vitro deacetylated, and analyzed, as described in Figure 2G. They were incubated with His-SIRT1 in the presence of 0.5 mM NAD+ or 5 mM NAM.
(C) HEK293T cells were cotransfected with a HIF-1α mutant, Flag-PCAF, and Myc/His-SIRT1, and subjected to 8 hr hypoxia.
(D) Recombinant GST-CT and GST-PCAF were purified using GSH-affinity beads, and incubated with recombinant His-SIRT1.
(E) HA-tagged HIF-1α or K674R was coexpressed with Flag-PCAF and Myc/His-SIRT1 in HEK293T cells subjected to 8 hr hypoxia.
(F) Sequence alignments of human, mouse, rat, and bovine HIF-1α proteins. The black bar denotes a conserved lysine residue acetylated/deacetylated by PCAF/SIRT1.
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6. Figure 4 HIF-1 Is Inactivated by SIRT1
(A) HT1080 cells were transfected with Myc/His-tagged SIRT1 or H363Y, and incubated for 16 hr. Acetylated HIF-1α was identified by immunoprecipitation and immunoblotting, and mRNAs were analyzed by RT-PCR and autoradiography.
(B) HT1080 cells were transfected with wild-type/mutated Epo-Luc, wild-type/mutated Myc/His-SIRT1, and β-gal, and incubated for 16 hr. Luciferase activities (means ± SD) were normalized to β-gal activity. ∗p < 0.01 (n = 8).
(C) HT1080 cells were transfected with wild-type/mutated Epo-Luc and 20 nM of SIRT1 siRNAs (I and II), and incubated for 16 hr. ∗p < 0.01 (n = 8).
(D) Epo-Luc-transfected HT1080 cells were treated with 25 μM resveratrol, and incubated for 16 hr. ∗p < 0.01 (n = 12).
(E) Epo-Luc and β-gal plasmids were cotransfected with wild-type or mutated HIF-1α (K674R; dN-TAD lacking aa 512–596; dC-TAD lacking aa 781–826) into HEK293T cells. Cells were incubated under normoxic or hypoxic conditions for 16 hr. Luciferase activities are represented as means ± SD (n = 8).
(F) Epo/mEPO-Luc or VEGF-Luc reporter gene was cotransfected with SIRT1 plasmid (or siRNA) and HIF-1α (or HIF-2α) in HEK293 cells. Luciferase activities are represented as means ± SD (n = 8). ∗p < 0.01 versus the HIF-1α control; #p < 0.01 versus the HIF-2α control.
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7. Figure 5 Expression and Activity of SIRT1 Decrease in Hypoxia
(A) SIRT1, HIF-1α, and PDK1 levels were analyzed in HT1080 cells incubated under hypoxic conditions for indicated times.
(B) SIRT1 and HIF-1α levels were analyzed in HEK293 and HCT116 cells, and the cells were subjected to 24 hr normoxia or hypoxia.
(C) In HT1080 cells subjected to 16 hr normoxia (N) or hypoxia (H), mRNA levels were quantified by real-time RT-PCR. Results (means ± SD) are represented as the relative levels versus 18S RNA. ∗p < 0.01 (n = 9) versus the normoxic group.
(D) HT1080 cells were transfected with 20 nM siRNA against HIF-1α, HIF-2α, and CtBP, and incubated under hypoxic conditions.
(E) HT1080 cells were transfected with CtBP siRNA, and incubated for 24 hr. Proteins and mRNAs were analyzed by immunoblotting and semiquantitative RT-PCR, respectively.
(F) In HEK293, HCT116, or HT1080 cells exposed to hypoxia for indicated time, NAD+ and total NAD levels were measured.
(G) HEK293T cells were transfected with HA-tagged, stable HIF-1α (sHIF-1α) and Flag-PCAF, and incubated for 16 hr. Acetylated sHIF-1α was identified by immunoprecipitation and immunoblotting.
(H) HEK293T cells were transfected with Flag-CT and CT/K674R, and incubated for 16 hr. Acetylated CTs were identified by immunoprecipitation and immunoblotting.
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8. Figure 6
The Glycolytic Pathway Positively Regulates HIF-1α Activity via SIRT1
(A) Hypoxia stimulates glycolysis but inhibits tricarboxylic acid cycle. Glycolysis inhibition by 2-deoxyglucose (2-DG) is known to block the conversion of NAD+ to NADH, which inactivates SIRT1.
(B) NAD+ and total NAD levels were measured in HT1080 cells subjected to 16 hr hypoxia with 1 or 5 mM 2-DG.
(C) Epo-Luc-transfected HT1080 cells were subjected to 16 hr hypoxia with 1 or 5 mM 2-DG. Luciferase activities (upper panel) are represented as means ± SD (n = 12). Cell lysates were immunoprecipitated with anti-p300 or anti-HIF-1α, and then p300-bound or acetylated HIF-1α was identified using anti-HIF-1α and anti-acetyl-lysine, respectively (lower panel).
(D) HT1080 cells were transfected with Epo-Luc and SIRT1 siRNA (I or II), and then subjected to 16 hr hypoxia with 5 mM 2-DG. Luciferase activities (top panel) are represented as means ± SD (n = 8). Acetylated HIF-1α was identified by immunoprecipitation and immunoblotting (middle panel), and mRNA levels were analyzed by RT-PCR and autoradiography (bottom panel).
(E) HEK293T cells, which had been cotransfected with EPO-Luc and HIF-1α (or K674R), were subjected to 16 hr hypoxia with 5 mM 2-DG. Luciferase activities are represented as means ± SD (n = 8).
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9. Figure 7 SIRT1-HIF-1α Interaction in Mouse Tissues and in Xenografts
(A–C) Mice were exposed to air (N) or 8% O2 (H) at 1 atmosphere for 4 to 24 hr, and tissues were excised and quickly frozen in liquid nitrogen. HIF-1α coprecipitated with SIRT1 or PCAF was identified using anti-HIF-1α (A). In brain tissues, SIRT1, NAD+, and total NAD levels were analyzed (B) and acetylated HIF-1α was identified (C).
(D) HT1080_vector and HT1080_M/H-SIRT1 cells were grafted on the left and the right flanks of nude mice, respectively (arrows). Volumes of HT1080_vector and HT1080_M/H-SIRT1 xenografts were monitored and the growth curves are plotted as means ± SEM (n = 10). Differences were compared using two-tailed, paired Student's t test. ∗p < 0.05; ∗∗p < 0.01 versus the vector group.
(E) Tumor tissues were fixed, embedded in paraffin, and cut for hematoxylin and eosin staining (H&E) and immunohistochemical staining. Representative H&E and CD31 sections are demonstrated in the left panel. Vascular distribution in tumors was estimated by counting CD31-positive filamentary structure (n = 10, means ± SD, two-tailed, unpaired t test).
(F) Tissue levels of VEGF were analyzed using an ELISA kit (n = 10; means ± SD, two-tailed, unpaired t test).
(G) Protein levels in tumor tissues were analyzed by immunoblotting, and acetylated HIF-1α levels by immunoprecipitation and immunoblotting. Each lane represents the result obtained from individual tumor.
Molecular Cell  , DOI: ( /j.molcel )
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