Figure 3. Relationships among IP3R1, NAC-inhibitable oxidation, CaMKII phosphorylation, and apoptosis. (a, left) Macrophages were transfected with four separate siRNA species directed against murine Ip3r1. 72 h after transfection, Ip3r1 mRNA was assayed by RT-QPCR. (right) A separate group of cells was subjected to control (Con) or cholesterol-loading conditions and then assayed for IICR (*, P < 0.05). (b, left) Macrophages treated as in panel a were assayed for apoptosis by annexin V staining (*, P < 0.05). (right) Another group of macrophages was preincubated for 1 h with vehicle control or 0.5 µM of the IP3R inhibitor xestospongin C (XestoC), subjected to control or cholesterol-loading (Chol) conditions, also in the absence or presence of xestospongin C, and then assayed for apoptosis (**, P < 0.01). (c) MEFs from WT and Ero1a−/− mice were incubated for 8 h with 5 mM azetidine (Aze) and then assayed for IICR (*, P < 0.05). (d, left) MEFs from WT and Ero1a−/− mice were incubated for 15 h with 10 µg/ml tunicamycin (TUN) or with 10 mM azetidine (AZE) and then assayed for apoptosis (*, P < 0.001 vs. WT MEFs; and **, P < 0.05 vs. tunicamycin group). (right) MEFs from WT mice were preincubated for 1 h with vehicle control or 0.5 µM xestospongin C, incubated for 15 h with 10 µg/ml tunicamycin or with 10 mM azetidine, also in the absence or presence of xestospongin C, and then assayed for apoptosis (*, P < 0.001 for Ero1a−/− vs. WT; for xestospongin C vs. untreated, and for azetidine vs. tunicamycin). (e) Macrophages were incubated for 8 h under control or cholesterol-loading conditions in the absence or presence of 1 mM NAC. The cells were then loaded with Fluo-3 and assayed for IICR (*, P < 0.001; and **, P < 0.05). (f) Macrophages were incubated for 16 h under control or cholesterol-loading conditions in the absence or presence of 1 mM NAC and then assayed for apoptosis. The bar graph shows the quantified data (*, P < 0.001). (a–f) Error bars show SEM (n = 3). (g) Macrophages from WT or Ero1a−/− mice were incubated for the indicated times under cholesterol-loading conditions. Cell lysates were then assayed by immunoblotting for expression of phospho-CaMKII (P-CaMKII), total CaMKII, ERO1-α, CHOP, and β-actin. Bar, 20 µm.
Figure 3.

Relationships among IP3R1, NAC-inhibitable oxidation, CaMKII phosphorylation, and apoptosis. (a, left) Macrophages were transfected with four separate siRNA species directed against murine Ip3r1. 72 h after transfection, Ip3r1 mRNA was assayed by RT-QPCR. (right) A separate group of cells was subjected to control (Con) or cholesterol-loading conditions and then assayed for IICR (*, P < 0.05). (b, left) Macrophages treated as in panel a were assayed for apoptosis by annexin V staining (*, P < 0.05). (right) Another group of macrophages was preincubated for 1 h with vehicle control or 0.5 µM of the IP3R inhibitor xestospongin C (XestoC), subjected to control or cholesterol-loading (Chol) conditions, also in the absence or presence of xestospongin C, and then assayed for apoptosis (**, P < 0.01). (c) MEFs from WT and Ero1a−/− mice were incubated for 8 h with 5 mM azetidine (Aze) and then assayed for IICR (*, P < 0.05). (d, left) MEFs from WT and Ero1a−/− mice were incubated for 15 h with 10 µg/ml tunicamycin (TUN) or with 10 mM azetidine (AZE) and then assayed for apoptosis (*, P < 0.001 vs. WT MEFs; and **, P < 0.05 vs. tunicamycin group). (right) MEFs from WT mice were preincubated for 1 h with vehicle control or 0.5 µM xestospongin C, incubated for 15 h with 10 µg/ml tunicamycin or with 10 mM azetidine, also in the absence or presence of xestospongin C, and then assayed for apoptosis (*, P < 0.001 for Ero1a−/− vs. WT; for xestospongin C vs. untreated, and for azetidine vs. tunicamycin). (e) Macrophages were incubated for 8 h under control or cholesterol-loading conditions in the absence or presence of 1 mM NAC. The cells were then loaded with Fluo-3 and assayed for IICR (*, P < 0.001; and **, P < 0.05). (f) Macrophages were incubated for 16 h under control or cholesterol-loading conditions in the absence or presence of 1 mM NAC and then assayed for apoptosis. The bar graph shows the quantified data (*, P < 0.001). (a–f) Error bars show SEM (n = 3). (g) Macrophages from WT or Ero1a−/− mice were incubated for the indicated times under cholesterol-loading conditions. Cell lysates were then assayed by immunoblotting for expression of phospho-CaMKII (P-CaMKII), total CaMKII, ERO1-α, CHOP, and β-actin. Bar, 20 µm.

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