JNCI Journal of the National Cancer Institute

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• Supplementary Fig. 1 - Sensitivity of ER stress– and DNA damage–induced Rat-1 cell death to Bcl-2 expression and caspase inhibition. A) Rat-1 cells stably transfected with the empty vector (Vector) or with vectors expressing Bcl-2 wt, Bcl-acta, and Bcl-cb5 were treated without or with the indicated death stimuli (tunicamycin, 70 μM; brefeldin A, 2 μg/mL; UV, 20 J/m2; etoposide, 20 μM) and assayed for apoptosis (upper panel) and cell death (lower panel) at 24 hours (18 hours for UV) after treatment. B) Rat-1 cells were treated without or with the indicated death stimuli as in (A), in the presence or absence of 150 μM Boc-Asp-fmk (BAF). The cells were then assayed for apoptosis (upper panel) and cell death (lower panel) at 21 hours (15 hours for UV) after treatment. In (A) and (B), the data represent the mean values of three independent experiments, and error bars correspond to the upper 95% confidence intervals.
• Supplementary Fig. 2 - Improved Rat-1 cell survival after ER stress and DNA damage caused by inhibition of oxidative phosphorylation. Rat-1 cells treated without or with 70 μM tunicamycin (left panel), 20 J/m2 UV (right panel) in the absence or presence of the indicated inhibitors were assayed for cell survival 48 hours after tunicamycin or UV treatment. The number of surviving cells relative to that of samples treated with tunicamycin or UV in the absence of inhibitors (Control, set to 1) is presented. The data represent the mean values of three independent experiments, and error bars correspond to the upper 95% confidence intervals. The concentrations of inhibitors used in this figure are as follows: rotenone, 0.1 μM; antimycin A, 7 μg/mL; oligomycin, 3 μg/mL; Trolox, 0.6 μM; Boc-Asp-fmk (BAF), 150 μM.
• Supplementary Fig. 3 - Specific cellular responses triggered by ER stress and DNA damage. Rat-1 cells treated without or with the indicated death stimuli (UV, 20 J/m2; etoposide, 20 μM; tunicamycin, 70 μM; brefeldin A, 2 μg/mL) were harvested 15 hours after treatment, and the whole-cell lysates were examined for protein levels of p53, Bip, CHOP, and β-actin (as loading control) by immunoblot analysis.
• Supplementary Fig. 4 - Suppression of cellular ATP level by inhibitors of the respiratory chain and oxidative phosphorylation. A) Rat-1 cells were cultured in the absence (Control) and presence of 0.1 μM rotenone, 7 μg/mL antimycin A, 3 μg/mL oligomycin, 0.6 μM Trolox, and 150 μM Boc-Asp-fmk (BAF) for 12 hours and then assayed for cellular ATP content. B) Rat-1 cells were cultured in the absence (Control) and presence of vehicle control for aurovertin B (chloroform, 0.1%), aurovertin B (20 μM, 10 μM, and 5 μM), and 3 μg/mL oligomycin. In (A) and (B), the results were normalized to the untreated sample (Control), which was set to 100%. The data represent the mean values of three independent experiments, and error bars correspond to the upper 95% confidence intervals.
• Supplementary Fig. 5 - Essential role for oxidative phosphorylation in oncogene-dependent Bax activation and cell death. A and B) Parental Rat-1 cells as well as Rat-1 cells stably expressing c-Myc (Rat-1/Myc, left panels) and RasV12 (Rat-1/Ras, right panels) were treated without or with 10 J/m2 UV and 40 μM tunicamycin, respectively, in the absence or presence of antimycin A (10 μg/mL) or oligomycin (3 μg/mL for parental Rat-1 cells, 6 μg/mL for Rat-1/Myc and Rat-1/Ras cells). A) Cells were harvested and analyzed for Bax oligomerization 4 hours after UV treatment (Rat-1/Myc, left panel) and 8 hours after tunicamycin treatment (Rat-1/Ras, right panel). B) Percentage of cell death was determined 6 hours after UV treatment (Rat-1/Myc, left panel) and 8 hours after tunicamycin treatment (Rat-1/Ras, right panel). The data represent the mean values of three independent experiments, and error bars correspond to the upper 95% confidence intervals.
• Supplementary Fig. 6 - Essential role for oxidative phosphorylation in ER stress–induced mitochondrial membrane permeabilization. Rat-1 cells were treated without or with 70 μM tunicamycin in the absence or presence of 7 μg/mL antimycin A or 3 μg/mL oligomycin for 12 hours. The cells were then harvested, and the cytosolic fractions of the cells were subjected to immunoblot analysis for cytochrome c. The membrane was reprobed for β-actin as loading control.
• Supplementary Fig. 7 - Oxidative phosphorylation–dependent activation of Bax and Bak, and cell death of HepG2 human hepatocellular carcinoma cells. HepG2 hepatocellular carcinoma cells were treated without or with 50 J/m2 UV in the absence or presence of the inhibitors (antimycin A, 0.6 μg/mL; oligomycin, 3 μg/mL; BAF, 150 μM; Trolox, 0.6 μ) (A). Alternatively, cells were treated without or with 50 μM etoposide in the absence or presence of the inhibitors (antimycin A, 0.6 μg/mL; oligomycin, 15 ng/mL; BAF, 150 μM; Trolox, 0.6 μM) (B). Then, cells were assayed for cell death (left panels) and Bax oligomerization (right panels) 8 hours after UV treatment and 14 hours after etoposide treatment. In (A) and (B), the data in the graphs represent the mean values of three independent experiments, and error bars correspond to the upper 95% confidence intervals.
• Supplementary Fig. 8 - Minimal cytosol-mitochondria translocation of Bax in Rat-1 cells after ER stress and DNA damage. Rat-1 cells treated without (-) or with (+) 70 μM tunicamycin or 20 J/m2 UV were harvested at 21 hours after tunicamycin treatment and 12 hours after UV treatment. Cells were then fractionated into cytosolic and mitochondrial fractions, which were then subjected to immunoblot analysis using polyclonal anti-Bax antibody (N-20).