Figure 4

Effect of ROS on macrophage MFG-E8 gene expression and efferocytosis. (A) ROS mimics the effect of alcohol on suppression of mRNA levels of MFG-E8 in macrophages. Peritoneal macrophages were subjected to treatment with culture medium alone (control), alcohol (25 mmol/L) or H₂O₂ (100 µmol/L) respectively for 18 h as indicated in the figure. After treatments, macrophages were processed for isolation of total cellular RNA followed by determining mRNA transcripts of MFG-E8 gene with a quantitative real-time RT-PCR method as described in the Figure 1 legend. (B) ROS mimics the effect of alcohol on suppression of MFG-E8 gene promoter activity. RAW 264.7 cells were dual transfected with plasmids of pRL-null and pGL3-mfge8^−444/+¹⁴⁶ reporters. Twenty-four hours after transfections, cells were then subjected to treatment with culture medium alone (control), alcohol (25 mmol/L) or H₂O₂ (100 µmol/L) for additional 18 h as indicated in the figure. Then, cells were processed for luciferase assay as described in the Figure 1 legend. (C) ROS mimics the effect of alcohol on impairment of macrophage efferocytosis. Peritoneal macrophages were subjected to treatment with culture medium alone (control), alcohol (25 mmol/L) or H₂O₂ (100 µmol/L) for 18 h as indicated in the figure. After treatments, macrophages were processed for appropriate staining followed by quantitatively determining macrophages that engulfed apoptotic cells using a flow cytometry—based method as described in the Figure 2 legend. Values are mean ± SEM and represent average of findings from two independent experiments with triplicate samples in each group. *P < 0.05 versus the control group.