GMFG modulates mtROS production and the mitochondrial respiration chain in macrophages. (A) Immunoblot analysis of GMFG, TfR1, and IRP1 in cellular lysates of RAW264.7 macrophages treated with hydrogen peroxide (0-250 µM) in 10% FBS/DMEM for 24 hours. α-tubulin was used as a loading control. (B-I) RAW264.7 macrophages were transfected with control siRNA (Ctrl) or GMFG siRNA for 48 hours, then stimulated without (M0) or with M1 (LPS/IFN-γ) or M2 (IL-4/IL-13) macrophage inducers for 24 hours. (B-E) mtROS, total mitochondrial mass, and mitochondrial membrane potential (Δψ_m) were analyzed by labeling cells with MitoSOX (B), MitoTracker Green (C), MitoTracker Red (D), or TMRM (E), respectively. Stained cells were then subjected to flow cytometry. (F-G) Immunoblot analysis of mitochondrial respiratory chain complex subunit proteins (complex I [CI; NDUFV2], complex II [CII; SDHD], complex III [CIII; CORE2], and complex IV [CIV; COX5A]) (F) or ISCU and antioxidant proteins (G). α-tubulin was used as a loading control. (H-I) Oxygen consumption rate (OCR) were measured under basal conditions followed by the sequential addition of oligomycin (1 µM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (0.5 µM), and rotenone (0.5 µM) plus antimycin A (0.5 µM) in control siRNA or GMFG siRNA-transfected Raw264.7 cells. Mean basal (H) and maximal (I) OCRs were measured using a Seahorse XF-24. OCRs were normalized by number of living cells in each condition. Basal OCR was measured over time for a single experiment. Data represent the mean ± standard deviation of at least 3 independent experiments. *P < .05 compared with control siRNA-transfected cells of the same phenotype.