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. 2010 Dec 13;191(6):1113–1125. doi: 10.1083/jcb.201006121

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© 2010 Li et al.

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Figure 4. — Evidence for CHOP amplification through NOX/oxidative stress–mediated activation of PKR. (A) Macrophages from WT or Nox2^−/− mice were incubated under cholesterol-loading conditions (CHOL) or with 7-ketocholesterol (7KC) for the indicated times. Lysates were then immunoblotted for phospho-CaMKII (p-CaMKII), total CaMKII (T-CaMKII), CHOP, phospho-PERK (p-PERK), phospho-PKR (p-PKR), and GAPDH. (B) Macrophages from WT or Nox2^−/− mice were incubated under cholesterol-loading conditions for 8 h (ATF4 experiment) or 9 h (XBP1 experiment). Nuclei were isolated and immunoblotted for ATF4 and nucleophosmin (Np) loading control, or RNA was extracted and assayed for spliced and unspliced Xbp1 and Gapdh loading control. (C) Macrophages were transfected with scrambled RNA (scrRNA) or Pkr siRNA. After 72 h, the cells were incubated for the indicated times under cholesterol-loading conditions. Lysates were then immunoblotted for PKR, phospho-eIF2α (P-eIF2α), CHOP, and β-actin loading control. (D) Macrophages were transfected with scrambled RNA or Pkr siRNA. After 72 h, the cells were incubated for an additional 8 h without sterol (Con) or with 7-ketocholesterol and then assayed for Nox2 mRNA. The data are displayed as Nox2 mRNA levels relative to those for the control scrambled RNA group. (E, left) Macrophages from WT, Chop^+/−, or Chop^−/− mice were incubated for 14 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol and then assayed for annexin V staining. (E, right) Macrophages were transfected with scrambled RNA or Pkr siRNA. After 72 h, the cells were incubated for an additional 30 h without sterol, under cholesterol-loading conditions, or with 7-ketocholesterol and then assayed for DCF fluorescence and annexin V staining. In this graph, * indicates P < 0.05 and ** indicates P < 0.01. (F) Peritoneal macrophages were pretreated in the absence or presence of 5 µM of the antioxidant N-acetylcysteine (NAC). The cells were then incubated under cholesterol-loading conditions or with 7-ketocholesterol for the indicated time periods, also in the absence or presence of NAC. Lysates were then immunoblotted for phospho-PKR, CHOP, and β-actin. n = 3 for each experimental group. Data are presented as means ± SEM.