Figure 1. Role of ERO1-α in ER stress–induced apoptosis in macrophages. (a) Macrophages from Chop+/+ (WT) and Chop−/− mice were incubated under control (Con) or cholesterol-loading (Chol) conditions for 8 h. Ero1a mRNA was then assayed by RT-QPCR (left; *, P < 0.001 for Ero1a mRNA). The graph on the top right shows the time course for this response; data points represent the mean of duplicate samples, which varied by <10% from each other. The immunoblot shows the ERO1-α protein level at 8 h. (b) Macrophages were transfected with three separate siRNA species directed against murine Ero1a. 72 h after transfection, ERO1-α protein was assayed by immunoblotting. (c) Macrophages were incubated for 30 h under control conditions, under cholesterol-loading conditions, or with 0.25 µM thapsigargin plus 25 µg/ml of the type A scavenger receptor ligand fucoidan. In each of these three groups of cells, there were four pretreatment subgroups: no RNA, scrambled (Scr) RNA, ERO1-α siRNA #2, or ERO1-α siRNA #3. At the end of the 30-h incubation, the cells were analyzed for apoptosis by annexin V staining (*, P < 0.01; and **, P < 0.05 vs. other values in same group). (d) Macrophages from WT or Ero1a−/− mice were assayed for apoptosis after incubation for 16 h under control conditions, under cholesterol-loading conditions, or with 50 µM 7-ketocholesterol (7-KC; *, P < 0.01; and **, P < 0.001 for Ero1a vs. WT macrophages). (e) Macrophages were pretreated with either scrambled RNA or ERO1-α siRNA #2, incubated for 15 h in the absence (Con) or presence of 100 nM staurosporine (STS), and then analyzed for apoptosis (*, P < 0.001 for STS vs. control). (f) Macrophages from WT or Chop−/− mice were transduced with adenovirus (Ad) containing murine Ero1a cDNA or a control LacZ construct at 500 MOI. 32 h after the addition of virus, one set of cells was harvested for ERO1-α immunoblotting, and another set was incubated for 16 h under control or cholesterol-loading conditions and then assayed for apoptosis (*, P < 0.001). (a and c–f) Error bars show SEM (n = 3).
Figure 1.

Role of ERO1-α in ER stress–induced apoptosis in macrophages. (a) Macrophages from Chop+/+ (WT) and Chop−/− mice were incubated under control (Con) or cholesterol-loading (Chol) conditions for 8 h. Ero1a mRNA was then assayed by RT-QPCR (left; *, P < 0.001 for Ero1a mRNA). The graph on the top right shows the time course for this response; data points represent the mean of duplicate samples, which varied by <10% from each other. The immunoblot shows the ERO1-α protein level at 8 h. (b) Macrophages were transfected with three separate siRNA species directed against murine Ero1a. 72 h after transfection, ERO1-α protein was assayed by immunoblotting. (c) Macrophages were incubated for 30 h under control conditions, under cholesterol-loading conditions, or with 0.25 µM thapsigargin plus 25 µg/ml of the type A scavenger receptor ligand fucoidan. In each of these three groups of cells, there were four pretreatment subgroups: no RNA, scrambled (Scr) RNA, ERO1-α siRNA #2, or ERO1-α siRNA #3. At the end of the 30-h incubation, the cells were analyzed for apoptosis by annexin V staining (*, P < 0.01; and **, P < 0.05 vs. other values in same group). (d) Macrophages from WT or Ero1a−/− mice were assayed for apoptosis after incubation for 16 h under control conditions, under cholesterol-loading conditions, or with 50 µM 7-ketocholesterol (7-KC; *, P < 0.01; and **, P < 0.001 for Ero1a vs. WT macrophages). (e) Macrophages were pretreated with either scrambled RNA or ERO1-α siRNA #2, incubated for 15 h in the absence (Con) or presence of 100 nM staurosporine (STS), and then analyzed for apoptosis (*, P < 0.001 for STS vs. control). (f) Macrophages from WT or Chop−/− mice were transduced with adenovirus (Ad) containing murine Ero1a cDNA or a control LacZ construct at 500 MOI. 32 h after the addition of virus, one set of cells was harvested for ERO1-α immunoblotting, and another set was incubated for 16 h under control or cholesterol-loading conditions and then assayed for apoptosis (*, P < 0.001). (a and c–f) Error bars show SEM (n = 3).

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