GMFG-knockdown modulation of iron metabolism protein expression and the M2 macrophage phenotype are associated with increased mtROS. (A) mtROS levels in RAW264.7 macrophages transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then treated with 8 mM of NAC for 30 minutes. Macrophages were then stained with MitoSOX and subjected to flow cytometry, and fluorescence-activated cell sorting MFI values were calculated. Intracellular ROS was expressed as the fold change of MFI normalized to the controls. Data represent the mean ± standard deviation of 3 independent experiments. (B) Immunoblot analysis of iron metabolism proteins in cellular lysates of RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 48 hours, then stimulated without (M0) or with M2 (IL-4/IL-13) macrophage inducers for 24 hours. Cells were subsequently treated with or without 8 mM of NAC for 30 minutes. α-tubulin was used as a loading control. (C-G) Quantitative polymerase chain reaction (qPCR) analysis of mean relative mRNA expression of M2 macrophage marker genes in RAW264.7 macrophages transfected with control siRNA or GMFG siRNA for 48 hours, then stimulated without (M0) or with M2 (IL-4/IL-13) macrophage inducers for 24 hours. Cells were subsequently treated with or without 8 mM of NAC for 30 minutes before RNA isolation. Expression levels were normalized to 18S mRNA expression. Data represent the mean ± standard deviation of at least 3 independent experiments. *P < .05 compared with no NAC treatment GMFG siRNA-transfected M2 cells.