1. Degradation of Misfolded Proteins Prevents ER-Derived Oxidative Stress and Cell Death 
Cole M. Haynes, Eric A. Titus, Antony A. Cooper 
Molecular Cell 
Volume 15, Issue 5, Pages (September 2004)
DOI: /j.molcel

2. Figure 1
HIP-Deficient Cells Are Sensitive to the Accumulation of Misfolded Proteins in the ER
(A) Wild-type (KHY163), hrd1Δ (KHY171), and erv29Δ (KHY270) cells expressing CPY*-1x and erv29Δ (KHY271) cells expressing wtCPY-1x were serially diluted, spotted onto plates, and grown at 30°C or 38°C.
(B) Western blot analysis of CPY* quantities in wild-type cells. 1x, CPY* expressed from the native promoter; 8x, CPY* expressed from the native promoter on a high copy (2 μ) plasmid; 25x, CPY* expressed from the CUP1 promoter in the presence of 150 μM CuSO4.
(C) A growth curve of wild-type, hrd1Δ, and erv29Δ cells expressing CPY*-25x (pAC595) following the addition of CuSO4.
(D) Wild-type and erv29Δ cells, expressing either CPY*-1x or CPY*-25x, were grown at 30°C or 38°C for 3 hr and UPR activation levels measured as described in Experimental Procedures. As a control for full UPR activation, wild-type cells were grown in 3 mM DTT for 3 hr.
(E) CPY*-25x expression was induced in wild-type and erv29Δ cells harboring pBG15 (CPY*-1x-HA, Caldwell et al., 2001). Cells were then harvested and fixed for indirect immunofluorescence at the times indicated. Cells were stained with anti-HA antibodies in combination with AlexaFluor 488 to detect CPY*-1x-HA, anti-Eug1p antibodies in combination with AlexaFluor 568 to detect the ER luminal marker, Eug1p, and DAPI to detect nuclei. Cell morphology was observed through differential interference contrast (DIC). At 4 hr, the fluorescence signal was overexposed to detect the diminishing signal.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
CPY*-25x Expression in HIP-Deficient Cells Leads to Cell Death that Is Dependent on UPR Activation
(A) A survivorship curve in which wild-type and erv29Δ cells were grown in liquid for the indicated amount of time (x axis) following induction of CPY*-25x expression. Cell survivorship was determined by growing wild-type (KHY163) and erv29Δ (KHY270) cells overnight in synthetic media to log phase (30°C) followed by the addition of 150 μM CuSO4 to induce expression of CPY*-25x (pAC595). To maintain wild-type cells in log phase, several dilutions were made throughout the time course. At the described times, 1 OD600nm was harvested, diluted 1:1000, and 300 μl of these cells were plated on synthetic media lacking CuSO4 and incubated at 30°C. Colony-forming units were then determined and a survivorship curve generated.
(B) UPR activation assays of wild-type and erv29Δ cells following induction of CPY*-25x expression. As a control for UPR activation, wild-type cells were grown in 3 mM DTT for 3 hr.
(C) A growth curve comparing wild-type, erv29Δ, erv29Δ ire1Δ (KHY438), and erv29Δ hac1Δ (KHY439) cells following induction of CPY*-25x expression.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
ER Stress Causes Cells to Display Some Hallmarks of Programmed Cell Death
TUNEL and annexin V assays (see Experimental Procedures) comparing wild-type (KHY163), erv29Δ (KHY270), erv29Δ ire1Δ (KHY438), and erv29Δ hac1Δ (KHY439) cells harboring pAC595 to express CPY*-25x.
(A) TUNEL assays to observe DNA fragmentation in wild-type and erv29Δ cells following induction of CPY*-25x for the indicated times. These cells did not appear to be necrotic as less than 5% of similarly treated cells did not stain with propidium iodide, indicating that the integrity of the plasma membrane was preserved (Madeo et al., 1997a).
(B) Annexin V assays (Madeo et al., 1997b) to examine PS externalization on the plasma membrane of wild-type and erv29Δ cells harvested following induction of CPY*-25x for the indicated times.
(C) DNA fragmentation in wild-type, erv29Δ, erv29Δ ire1Δ, and erv29Δ hac1Δ cells following induction of CPY*-25x. Cells were harvested at the indicated time points and TUNEL assays performed. At least 300 cells were counted to determine the percentage of TUNEL-positive cells at each time point.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
ER Stress and Prolonged UPR Activation Leads to ROS Accumulation
(A) Fluorescence microscopy comparing DH123 stained wild-type (KHY163) and erv29Δ (KHY270) cells following expression of CPY*-25x for the indicated amount of time. Cells were stained with DH123 for 2 hr then visualized using filter sets specific for rhodamine and DIC to observe cell morphology.
(B) Fluorescence microscopy comparing DH123 stained erv29Δ, erv29Δ ire1Δ (KHY438), and erv29Δ hac1Δ (KHY439) following expression of CPY*-25x for 2 hr.
(C) A growth curve comparing erv29Δ cells expressing CPY*-25x incubated with 0 or 1 mM GSH. GSH was added 1 hr after induction of CPY*-25x.
(D) Fluorescence microscopy of DH123 stained erv29Δ cells following addition of 1 mM GSH. The cells were induced to express CPY*-25x, an aliquot of cells was harvested at 1 hr, and 1 mM GSH was added to the remaining cells which were harvested 2 hr later and stained with DH123.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Oxidative Protein Folding in the ER Contributes to ROS Accumulation and Cell Death
(A) A depiction of disulfide bond oxidation and reduction in the lumen of the ER indicating the flow of electrons and oxidizing equivalents (Cuozzo and Kaiser, 1999).
(B) Flow cytometry analysis of DH123-stained erv29Δ ire1Δ (KHY438) cells expressing CPY*-25x (pAC595) in the presence (black line) or absence (gray line) of ERO1 overexpression (pAC672). Flow cytometry was performed as described (Madeo et al., 1999).
(C) Growth curves comparing wild-type (KHY298) and erv29Δ (KHY318) cells expressing CPY*-25x or cysteine-less CPY*-25x (pAC774).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Oxidation of Glutathione Is Associated with ER Stress and Prolonged Interactions between CPY* and Pdi1p
(A) Quantification of GSSG/GSH following induction of CPY*-25x in erv29Δ (KHY270) cells compared to wild-type cells (SEY6211a). Quantification was performed as described (Cuozzo and Kaiser, 1999).
(B) Autoradiograph and quantification of Pdi1p interactions with either wtCPY-25x or CPY*-25x via mixed disulfide bond. erv29Δ cells containing pAC595 or erv29Δ (KHY271) containing pAC667 were incubated in 150 μM CuSO4 for 30 min followed by radiolabeling for 10 min, chase for 2 min, cycloheximide (50 μg/ml) addition for 2 min, and cells were harvested at the described time points. The top panel shows the amount of CPY* or wtCPY bound to Pdi1p via a mixed disulfide. Isolation and quantification of mixed disulfides was performed as described (Frand and Kaiser, 1999) (see Experimental Procedures). In short, cells were harvested, suspended in 10% TCA to block thiol exchange, lysed in a buffer containing SDS under nonreducing conditions, divided into four aliquots, and Pdi1p was immunoprecipitated under nonreducing conditions from one of the aliquots. The samples were then exposed to 100 mM DTT to break disulfide bonds, diluted, and incubated with anti-CPY antibodies to immunoprecipitate the wtCPY/CPY* that interacted with Pdi1p via mixed disulfide bond. The samples were run on a reducing 10% SDS page gel. The remaining three aliquots which represent 1/7 the cell lysate used in the top panel were immunoprecipitated under reducing conditions for the indicated antigens, and they represent the total relative amount of each protein. Vma1p was used as a loading/lysis control. The graph represents the percentage of wtCPY or CPY* in a cell that is bound to Pdi1p via mixed disulfide bond at the indicated time following the chase.
(C) Fluorescence microscopy comparing DH123 staining in erv29Δ cells expressing CPY*-25x and erv29Δ cells expressing wtCPY-25x for 2 hr.
(D) Growth curves comparing CPY*-25x expression to that of wtCPY-25x in wild-type (SEY6211a) and erv29Δ (KHY271) cells. The PRC1 ORF encoding wtCPY was inserted downstream of the CUP1 promoter so the expression level would be identical to that of CPY*-25x (data not shown).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Respiring Mitochondria Contribute to ROS Accumulation and Cell Death during ER Stress
All cells were grown in synthetic media containing 2% glycerol and 0.5% glucose.
(A) Fluorescence microscopy of ρ0-derived wild-type (KHY163) and ρ0-derived erv29Δ (KHY270) cells stained with dihydroethidium for 10 min following 1.5 hr of CPY*-25x (pAC595) expression (Madeo et al., 1999).
(B) Growth curves of ρ0-derived wild-type and ρ0-derived erv29Δ cells following induction of CPY*-25x expression.
(C) TUNEL assays comparing ρ0-derived wild-type and ρ0-derived erv29Δ cells following CPY*-25x expression for 5 hr.
Molecular Cell  , DOI: ( /j.molcel )