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

Figure 1.

Figure 1.

ER-stressed macrophages undergo oxidative stress, which is dependent on CHOP, calcium, and CaMKII. (A) Peritoneal macrophages from Chop+/+ (WT) and Chop−/− mice were incubated for 15 h without sterol (Con), under cholesterol-loading conditions (CHOL), or with 7-ketocholesterol (7KC). Intracellular peroxide accumulation was then assayed by DCF fluorescence. Three fields for each sample were quantified and expressed as a percentage of DCF-positive cells. (B) Macrophages were pretreated for 1 h with 5 µM BAPTA-AM or with equivalent volumes of vehicle (Veh). The cells were then incubated for 15 h under control or cholesterol-loading conditions and also with BAPTA-AM or vehicle control as indicated. Intracellular peroxide accumulation and apoptosis were assayed by DCF fluorescence (green) and Alexa Fluor 594–conjugated annexin V (red), respectively. Bar, 20 µm. (C) Macrophages were pretreated for 1 h in the absence or presence of 5 µM of the CaMKII inhibitor KN93 or the inactive analogue KN92, followed by incubation for 14 h without sterol under cholesterol-loading conditions or with 7-ketocholesterol. DCF fluorescence and annexin V staining were then assayed. (D) Macrophages were transfected with two different Camk2g siRNA constructs. After 72 h, the cells were incubated for 30 h under control or cholesterol-loading conditions and then assayed for DCF fluorescence and annexin V staining. Bars with the same symbols are not significantly different from each other, whereas bars with different symbols are significantly different from each other. n = 3 for each experimental group. scrRNA, scrambled RNA. Data are presented as means ± SEM.