Skip to main content
. 2014 Sep;86(3):284–296. doi: 10.1124/mol.114.093039

Fig. 1.

Fig. 1.

Effects of globular adiponectin on ethanol-induced ROS production and NADPH oxidase activation in macrophages. (A) RAW 264.7 macrophages cultured in four-well culture slides were pretreated with gAcrp (0.1 μg/ml) for 18 hours, followed by stimulation with 100 mM ethanol for an additional 24 hours. Cells were then further stimulated with HBSS containing 10 μM of CM-H2DCF-DA; ROS production was assessed by conversion of CM-H2DCF-DA to the oxidized fluorescent DCF, which was monitored by fluorescence microscopy as described in Materials and Methods. Representative images from three independent experiments are shown along with quantification with green signal intensity (lower panel). Values are expressed as mean ± S.E.M. (n = 3). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol. (B) Peritoneal macrophages isolated from mice were incubated with 0.1 μg/ml of gAcrp for 18 hours, followed by treatment with 100 mM ethanol for an additional 24 hours. ROS production was assessed as described previously, and representative images from three independent experiments are shown along with the quantitative analysis of green fluorescent intensity (lower panel). Values shown are presented as mean ± S.E.M. (n = 3). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol. (C) RAW 264.7 macrophages were treated with 0.1 μg/ml of gAcrp for 18 hours, followed by incubation with 100 mM ethanol for 24 hours. NADPH oxidase activity was determined by lucigenin-based assay as described in Materials and Methods. Briefly, cell lysates were incubated with HBSS containing 100 μM lucigenin and 200 μM NADPH for 30 minutes. NADPH oxidase activity was then assessed by chemiluminescence and expressed in relative light units. Values represent fold increase in comparison with cells not treated with ethanol and are expressed as mean ± S.E.M. (n = 6). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol. (D) Primary murine peritoneal macrophages were pretreated with gAcrp (0.1 μg/ml) for 18 hours followed by incubation with ethanol (100 mM) for an additional 24 hours. NADPH oxidase activity was measured as described previously. Values are presented as fold increase compared with the control cells and are expressed as mean ± S.E.M. (n = 4). *P < 0.05 compared with cells not treated with ethanol; #P < 0.05 compared with cells treated with ethanol.