The effect of iron and lipopolysaccharide (LPS) on mitochondrial reactive oxygen species production. (A) Effect of iron supplementation on mitochondrial reactive oxygen species production. The shaded histogram depicts unstained cells. The histogram in red shows the effects of antimycin (20 μg/ml), an inhibitor of mitochondrial complex III used as a positive control. Iron-naïve cells treated with lipopolysaccharide do not show increased MitoSOX fluorescence compared to controls (middle). However, iron-pretreated RAW cells exposed to lipopolysaccharide have more mitochondrial reactive oxygen species production compared to cells treated with lipopolysaccharide alone (right), although the increase is similar to that caused by iron incubation alone (left). Data are representative of three experiments. (B) Summary bar graphs showing relative MitoSOX fluorescence (two-way ANOVA; interaction between iron and lipopolysaccharide not significant, therefore only main effects reported; n = 3 replicates/condition). (C, D) Incubating RAW cells with 100 μM of the mitochondrion-specific antioxidant MitoTEMPO blunted the mRNA response to lipopolysaccharide (Il6 and Mcp1) in iron-loaded macrophages (unpaired t tests; n = 3 replicates/condition). Il = interleukin; Int = interaction P value; Med = medium; MFI = mean fluorescent intensity.