FIGURE 3: Effects of iron perturbations on TCA enzyme expression and metabolite concentrations in Salmonella infected macrophages. Cells were pre-treated with FeCl₃/hepcidin (50 μM/ 1μg/ml, respectively) or DFP (50 μM) for 6 hours or left untreated prior to infection with Salmonella (MOI of 10) as detailed in methods. Expression of aconitase (A), isocitrate dehydrogenase (B), succinate dehydrogenase (C) and lactate dehydrogenase (D) mRNA relative to the house keeping gene was determined by qRT-PCR in untreated control cells (ctrl) or infected cells after 24 hours. Metabolite levels of lactate (E), free fatty acids (F) and pyruvate (G) were determined in the cell supernatant after 24 h of infection. A selection of 15-top metabolites (raw-wise) from the metabolomics analysis of cellular supernatants is shown in the heatmap (H), where samples in each group were averaged (column-wise, S.Tm/Fe n=13; S.Tm n=13). Representative data from three independent experiments performed in duplicates or triplicates are shown. Data are shown as relative changes as compared to the control. Graphs show means ± SEM. One-way ANOVA with Tukey's multiple comparison correction was performed. Exact p-values are indicated in the graphs.