1. Volume 11, Issue 3, Pages 619-633 (March 2003)
An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress 
Heather P. Harding, Yuhong Zhang, Huiquing Zeng, Isabel Novoa, Phoebe D. Lu, Marcella Calfon, Navid Sadri, Chi Yun, Brian Popko, Richard Paules, David F. Stojdl, John C. Bell, Thore Hettmann, Jeffrey M. Leiden, David Ron 
Molecular Cell 
Volume 11, Issue 3, Pages (March 2003)
DOI: /S (03)
Copyright © 2003 Cell Press Terms and Conditions

2. Figure 1 Impaired Amino Acid Metabolism in Atf4−/− Cells
(A) Immunoblots of endogenous PERK and IRE1α immunoprecipitated from detergent lysates and ATF4 and CHOP from the nuclear fractions of the same untreated and thapsigargin-treated (Tg, 0.4 μM) Atf4+/+ and Atf4−/− mouse fibroblasts. Activation of PERK and IRE1α is revealed by reduced mobility of their phosphorylated forms (P-PERK and P-IRE1α) compared to the inactive underphosphorylated forms (PERK0 and IRE1α0).
(B) Northern blot analysis of the glycine transporter 1 (Glyt1), heavy chain of the X−c cystine/glutamate exchanger (Slc3a2), and asparagine synthase (Asns) genes in untreated cells with the indicated genotypes (UT), cells deprived of leucine for 4 and 8 hr (L4 and L8), or cells treated with the ER stress-inducing agent tunicamycin for 8 hr (Tm, 2 μg/ml).
(C) Photomicrograph of crystal violet-stained cells grown in media with and without amino acid supplementation (NEAA).
(D) Growth curves of cells described in (C).
(E) Immunoblot of activated GCN2 (P-GCN2), total GCN2, phosphorylated eIF2α, and total eIF2α from cells cultured in complete media with or without supplemental amino acids (NEAA) or in media lacking leucine (Leu−).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

3. Figure 2
List of Genes Induced at Least 2-Fold by Tunicamycin in Wild-Type Cells (p ≤ 0.05)
“Fold induction” is the mean ± SD of the ratio of the hybridization signals in tunicamycin-treated versus untreated cells (Tm/UT±SD). The measurements are color coded as indicated under the figure. “Expression Level” is defined as the ratio of the average signal strength of each gene in each experimental group to the median signal strength of that gene derived from all experimental groups. The expression level of each gene in tunicamycin-treated Atf4−/− and Perk−/− is presented as a percentage of its expression level in tunicamycin-treated wild-type cells (% of WT±SD). The measurements are color coded as indicated under the figure.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

4. Figure 3 Atf4−/− Cells Require Supplemental Reducing Compounds
(A) Photomicrograph of crystal violet-stained cells grown in media lacking supplements (−) or containing reduced glutathione (GSH), N-acetyl cysteine (NAC), or β-mercaptoethanol (βME) with and without amino acid supplementation (NEAA).
(B) Survival, defined by the ability to reduce MTT, of Atf4+/+ (dotted line) and Atf4−/− cells (solid line) 36 hr after switching from media containing 55 μM β-mercaptoethanol into media containing the indicated supplements. 100% survival is defined as MTT reduction by wild-type cells that had remained in 55 μM β-mercaptoethanol. Shown are mean and SEM of an experiment performed in duplicate and reproduced twice.
(C) Death, defined by permeability to propidium iodide, 24 hr after cells were switched from media containing 55 μM β-mercaptoethanol into media containing the indicated supplements. Shown are mean ± SEM (n = 2).
(D) Glutathione levels in cells growing in media containing the indicated concentrations of β-mercaptoethanol (βME) or cysteine (Cys). Shown are mean ± SEM (n = 2).
(E) FACS measurement of Dichlorofluorescein (DCF) fluorescence of cells maintained in 55 μM β-mercaptoethanol or switched to media containing no additives, 1 mM cysteine, or 1 mM cystine for 24 hr.
(F) Effect of 10 μM desferoxamine (DF) on survival of wild-type and Atf4−/− cells cultured in the presence and absence of β-mercaptoethanol. 100% survival is defined as the MTT activity of the cells maintained in media supplemented with β-mercaptoethanol but no desferoxamine.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

5. Figure 4 Endogenous Peroxide Accumulation in Perk−/− Cells
(A) FACS measurement of Dichlorofluorescein (DCF) fluorescence in cells following exposure to tunicamycin (0.25 μg/ml).
(B) Fluorescent photomicrographs of tunicamycin-treated Perk−/− cells immunostained with an antibody to Cytochromec (green). Note that loss of mitochondrial localization of Cytochromec fluorescence (arrows) is a late event compared with the acquisition of DCF fluorescence as shown in (A).
(C) Plot of DCF fluorescence in Perk−/− cells treated with tunicamycin in the presence or absence of the caspase inhibitor zVAD (20 μM). Shown are mean ± SEM (n = 2).
(D) Immunoblot detecting caspase 9 cleavage in wild-type and Perk−/− cells treated with tunicamycin in the presence or absence of the caspase inhibitor zVAD.
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

6. Figure 5
Oxidative Stress Contributes to Death of ER-Stressed Perk−/− Cells
(A) FACS measurement of Dichlorofluorescein (DCF) fluorescence following exposure to tunicamycin (0.25 μg/ml, 16 hr) in the presence or absence of Cystine.
(B) Percentage of dead cells (propidium iodide positive) following 16 hr of culture in the presence or absence of tunicamycin (0.25 μg/μl), β-mercaptoethanol (βME, 55 μM), cysteine (C, 1 mM), or cystine (C-C, 1 mM). Shown are mean ± SEM (n = 4).
(C) Survival at 24 hr of Perk−/− cells exposed to low dose tunicamycin (60 ng/ml) in the presence or absence of desferoxamine (DF). Shown are mean ± SEM (n = 4).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions

7. Figure 6
Inhibition of ero-1 Blocks Endogenous Peroxide Accumulation and Protects PERK (pek-1)-Deleted C. elegans from Premature Death
(A) Fluorescent micrographs of hsp-4::gfp(zcIs4)V (an ER stress-inducible reporter), [ero-1(RNAi)I; hsp-4::gfp(zcIs4)V], [ero-1(RNAi)I; ire-1(zv14)III; hsp-4::gfp(zcIs4)V], and tunicamycin-treated (Tm) hsp-4::gfp(zcIs4)V animals.
(B) Dichlorofluorescein fluorescent micrographs of wild-type (WT) and pek-1(zcDf2)X animals, exposed to tunicamycin and subjected to ero-1(RNAi).
(C) Survival curves of wild-type, ero-1(RNAi)I, pek-1(zcDf2)X, and [ero-1(RNAi)I; pek-1(zcDf2)X] animals. Shown is the average number of survivors from two cohorts of 100 animals each. Data pairs of pek-1(zcDf2)X and [ero-1(RNAi)I, pek-1(zcDf2)X] with a significant survival difference are indicated with an asterisk (p < 0.05).
Molecular Cell  , DOI: ( /S (03) )
Copyright © 2003 Cell Press Terms and Conditions