1. Volume 42, Issue 5, Pages 597-609 (June 2011)
A HIF-1 Target, ATIA, Protects Cells from Apoptosis by Modulating the Mitochondrial Thioredoxin, TRX2 
Swati Choksi, Yong Lin, Yelena Pobezinskaya, Li Chen, Chung Park, Michael Morgan, Tao Li, Siriporn Jitkaew, Xiumei Cao, You-Sun Kim, Hong-Sug Kim, Peter Levitt, Grace Shih, Michael Birre, Chu-Xia Deng, Zheng-gang Liu 
Molecular Cell 
Volume 42, Issue 5, Pages (June 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 Schematic Presentation and Expression of ATIA
(A) N-terminal leucine-rich repeat (LRR), an EGF signature, a fibronectin3 domain (FN3), and a C-terminal transmembrane domain are highlighted.
(B) Northern blot analysis of ATIA mRNA expression in different tissues.
(C) ATIA expression is downregulated in TRAF2−/− cells. Total RNA was isolated from WT, TRAF2−/−, and TRAF2 reconstituted MEFs and analyzed by northern blot.
(D) ATIA is not induced by TNFα treatment. WT MEF cells were treated with TNFα for the indicated times, and total RNA was isolated. ATIA mRNA was detected by northern blot.
(E) Both ATIA full-length and ATIAc protect cells from TNFα-induced apoptosis.
TRAF2−/− MEFs were transiently transfected with YFP-tagged ATIA full-length, ATIAc, and vector alone and treated with TNFα and CHX for 5 hr. The percent YFP-positive and PI negative cells for each treatment group was determined by FACS. ∗p < 0.05 versus vector-YFP control. Error bars represent the standard error of the mean (SEM). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Subcellular Localization and Protective Role of Full-Length and Truncated ATIA
(A) Western blot analysis of the expression of ATIA-YFP and ATIAc-YFP transiently transfected into WT MEFs. The cell lysates were immunoblotted and analyzed with the indicated antibodies.
(B) Confocal microscopy of live WT MEFs transiently transfected with ATIA-YFP and ATIAc-YFP constructs. Mitochondrial staining was performed by MitoTracker.
(C) Western blot analysis of pronase-treated ATIA-YFP- or ATIAc-YFP-transfected MEFs. The cells lysates were immunoblotted with the indicated antibodies.
(D) Western blot analysis of endogenous ATIA after pronase treatment of MEFs followed by cellular fractionation. The pronase treated and untreated cytosolic (C) and mitochondrial (M) fractions and the total cell lysates (T) were blotted with the indicated antibodies. AIF is a mitochondrial specific control, whereas HSP90 is cytosolic specific. TNFR1 is a control for membrane specific proteins. The ∗ indicates a nonspecific band.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Schematic Presentation of Truncated Forms of ATIA and Their Localization
(A) The various truncated forms of ATIA with loss of specific domains are shown.
(B and C) Confocal microscopy of live WT MEFs transiently transfected with various constructs. Mitochondrial staining was performed by MitoTracker.
(D) ATIA−/− MEFs were transiently transfected with YFP-tagged ATIA vector alone, full-length, 287–673, or 409–673. Comparable expression of these plasmids was determined by western blot analysis with anti-GFP antibody (right panel). The transfected cells were treated with TNFα and CHX for 8 hr (left panel). The percent YFP-positive and PI negative cells for each treatment group was determined by FACS. ∗p < versus control with vector. Error bars represent the SEM.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Hypoxia Induces ATIA Expression
(A and B) Western blot analysis of WT MEFs treated with CoCl2 (A) or physiological hypoxia (B) for the indicated times. The cell lysates were immunoblotted with the indicated antibodies.
(C and D) Real-time PCR of total RNA isolated from WT MEFs treated with CoCl2 (C) or 0.1% hypoxia (D) for the indicated times. For (C), ∗p < 0.04 versus untreated control; for (D), ∗p < 0.03 versus untreated control. Error bars represent the SEM.
(E) ChIP Analysis of WT MEFs treated with 0.1% hypoxia for 8 hr. Chromatin immunoprecipitation was done with anti-HIF-1α antibody and goat IgG isotype control.
(F) Western blot analysis after CoCl2 treatment of WT MEFs transfected with 50 pMol nontargeting (NT) or HIF-1α (pool#1) siRNA. The cells lysates were immunoblotted with the indicated antibodies.
(G) Western blot analysis after 0.1% hypoxia treatment of WT MEFs transfected with 50 pMol nontargeting (NT) or HIF-1α (pool#2) siRNA. The cell lysates were immunoblotted with the indicated antibodies.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 ATIA Protects from TNFα- and Hypoxia-Induced Apoptosis
(A) ATIA+/+ and ATIA−/− mice were injected with GalN and TNFα. Survival curves of ATIA+/+ (n = 5) and ATIA−/− (n = 8) after treatment.
(B) TUNEL staining of liver tissue isolated from ATIA+/+ and ATIA−/− mice 4 hr after administration of GalN alone or GalN and TNFα. Increased apoptosis is seen in GalN and TNFα treated ATIA−/− as compared to the ATIA+/+ liver tissue (left panel). The level of TUNEL staining is quantified to show increased apoptosis in ATIA−/− livers as compared to wild-type when treated with TNFα and GalN for 4 hr (right panel).
(C) Representative TUNEL staining of testis tissue sections from 4-week-old ATIA+/+ and ATIA−/− mice (left panels). Arrows indicated TUNEL-positive cells. Apoptosis in the testis of different mice as indicated was quantified by the number of apoptotic cells per 100 tubules (right panel). Data shown is the average of three mice for each group. ∗p < 0.03 as compared to WT mice.
(D) ATIA−/− MEFs are more sensitive to TNFα-induced apoptosis. ATIA+/+ and ATIA−/− MEFs were treated with TNFα and CHX for the indicated times. Cell survival was measured by MTT assay and was calculated by comparison of the TC treatment group to the CHX treatment group.
(E) ATIA−/− MEFs were reconstituted with Vector-YFP, ATIA-YFP, or ATIAc-YFP and treated with TNFα and CHX for 12 hr. Propidium iodide staining was used to determine cell viability. The percent YFP-positive cells for each treatment group was determined by FACS. ∗p < as compared to with vector-YFP.
(F) ATIA+/+ and ATIA−/− MEFs were treated with CoCl2 for the indicated times. Cell survival was measured by MTT assay. Survival ratio was calculated by comparing each treatment group to untreated cells. ∗p < as compared to untreated control.
(G) ATIA+/+ and ATIA−/− MEFs were treated with physiological hypoxia. Cell survival was measured by FACS analysis of annexin-positive and PI-negative cells for each treatment group. Survival ratio was calculated by comparison of each treatment group to untreated cells. ∗p < as compared to untreated control.
Error bars represent the SEM. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 ATIA Modulates TRX2 Oxidization and ROS Production
(A) In vitro GST pull-down assay. ATIA-YFP was transfected into WT MEFs. Cell lysates were applied to GST- and GST-TRX2 beads in lysis buffer. Bound ATIA protein was detected by Western blot with anti-GFP.
(B–D) Different TRX2 redox states in ATIA+/+ and ATIA−/− MEF cells. ATIA+/+ and ATIA−/− MEF cells were treated with H2O2 (B), TNFα and CHX (C), or CoCl2 (D). Separation of reduced and oxidized TRX2 bands is performed by AMS alkylation of thiols followed by SDS-PAGE.
(E and F) ATIA+/+ and ATIA−/− MEFs were treated with TNFα and CHX or CoCl2 for the indicated times, stained with DCFDA, and analyzed by flow cytometry. Grey histograms represent background level of ROS with no treatment.
(G) ATIA+/+ and ATIA−/− MEFs were treated with TNFα and CHX in the presence of BHA (100 mM) or NAC (2.5 mM) for 10 hr. Cell survival was measured by MTT assay and was calculated by comparison of the TC treatment group to the CHX and BHA or NAC treatment group. ∗p < as compared to cells treated with just TNFα and CHX.
(H) ATIA+/+ and ATIA−/− MEFs were treated with CoCl2 for 16 hr. Cell survival was measured by MTT assay. Survival ratio was calculated by comparison of each treatment group to untreated cells. ∗p < as compared to CoCl2-treated cells.
Error bars represent the SEM. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 ATIA Is Highly Expressed in Human Glioblastomas
(A) ATIA protein was detected in brain tumor by western blotting.
(B) Western blotting of human brain tumor cell lines with the indicated antibodies.
(C) Sections of human glioblastoma samples stained with H&E and anti-vasorin antibody.
(D) Brain tumor tissue arrays with normal brain tissue controls were immunostained with anti-vasorin antibody. The data from three different brain tumor arrays was compiled, and the percentage of ATIA-positive cases was determined.
(E and F) A-172 cells were transfected with 50 pMol non-targeting (NT) or ATIA siRNA and 24 hr later were treated with CoCl2 or hypoxia for the indicated times. Cell survival was measured by MTT assay (E) or by FACS of annexin-positive and PI negative cells for each treatment group (F). Survival ratio was calculated by comparison of each treatment group to untreated cells. ∗p < (E) or ∗p < 0.01 (F) as compared to untreated control.
Error bars represent the SEM. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions