Fpn1 expression and effects of DFX on effector functions in Nos2^+/+ and Nos2^−/− PE-MΦ. (A and B) Nos2 wild-type and Nos2^−/− PE-MΦ were isolated and treated with PBS or infected with S. typhimurium (S. tm.). (A) Fpn1 in comparison to actin protein expression was determined by means of Western blotting. Data from one of four independent experiments are shown. (B) Fpn1 and actin levels were quantified by densitometric scanning of membranes. Data were compared by ANOVA using Bonferroni’s correction (n = 8 independent experiments). (C) PE-MΦ were transiently transfected with the 8.4-kb Fpn1 promoter firefly luciferase construct and pRL-SV40 Renilla luciferase plasmid and infected with S. typhimurium. Relative chemiluminescence values are depicted (n = 4 independent experiments). (D) ⁵⁹Fe release in response to S. typhimurium infection was determined after loading of infected macrophages with 10 µM ⁵⁹Fe-citrate. Data were compared and are depicted as in Fig. 2 D (n = 6 independent experiments). (E) Nos2^+/+ and Nos2^−/− PE-MΦ were infected and treated with PBS or 50 µM of the iron chelator DFX. Intracellular bacterial load was determined by plating (n = 5–6 independent experiments). (F and G) Fpn1 expression and iron content in RAW264.7 cells expressing (Nramp1^R) or lacking functional Nramp1 (Nramp1^S) was determined after infection with S. typhimurium and stimulation with 10 ng/ml IFN-γ or 600 µM L-NIL. (F) Fpn1 expression was determined by qRT-PCR and data were normalized for mRNA levels of Hprt (n = 6 independent experiments). (G) Cellular iron content of RAW264.7 Nramp1^R-expressing cells as compared with RAW264.7 Nramp1^S-expressing cells was measured by atomic absorption spectrometry. Results were normalized for protein content (n = 12 independent experiments). ^#, P < 0.10; *, P < 0.05; **, P < 0.01; ***, P < 0.001.