Figure 2. Fpn1 expression, macrophage iron release, and bacterial iron acquisition after pharmacological blockade of NO formation. (A–C) Nos2+/+ PE-MΦ were infected with Salmonella typhimurium (S. tm.) and treated with PBS or 600 µM of the NOS2 inhibitor L-NIL for 24 h. (A) Fpn1 expression was determined by qRT-PCR, and data were normalized for mRNA levels of Hprt and compared by ANOVA using Bonferroni’s correction for multiple tests. Values are depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges and statistically significant differences are indicated (n = 4 independent experiments). (B) Fpn1 and actin protein levels were assessed by Western blotting, with one of four independent experiments shown. (C) Fpn1 and actin levels were quantified by densitometric scanning of membranes (n = 8 independent experiments). (D–F) 59Fe transport studies were used to determine macrophage iron release and bacterial iron acquisition within macrophages and in liquid culture, respectively. (D) After loading with 10 µM 59Fe-citrate, macrophage iron release was determined (n = 6 independent experiments). (E) Bacterial iron acquisition within macrophages was determined after loading of infected macrophages with 10 µM 59Fe-citrate. Data were normalized for Salmonella numbers as determined by plate counting (n = 6 independent experiments). (F) Bacterial iron acquisition in liquid culture was determined after incubation with 10 µM 59Fe-citrate. As control, an isogenic triple mutant, deficient in enterobactin synthesis, sitABCD-, and feo-mediated iron uptake (S. tm. entC sit feo) was used. Data are depicted relative to the solvent-treated control (Ctrl.) as described in A (n = 4 independent experiments). (G) Wild-type PE-MΦ were infected with Salmonella typhimurium (S. tm.) and Nrf2-binding activity was determined by a commercially available assay and depicted as arbitrary units (AU; n = 8–10 independent experiments). (H) Wild-type PE-MΦ were transiently transfected with an 8.4-kb Fpn1 promoter luciferase construct (Fpn1 8.4-kb), a variant carrying a specific mutation in the Nrf2-binding site (Nrf2 mut.), or empty plasmid (not depicted), and subsequently treated with PBS or infected with S. typhimurium (S. tm.). Luciferase activity is expressed as arbitrary light units (ALU; n = 4 independent experiments). **, P < 0.01; ***, P < 0.001.
Figure 2.

Fpn1 expression, macrophage iron release, and bacterial iron acquisition after pharmacological blockade of NO formation. (A–C) Nos2+/+ PE-MΦ were infected with Salmonella typhimurium (S. tm.) and treated with PBS or 600 µM of the NOS2 inhibitor L-NIL for 24 h. (A) Fpn1 expression was determined by qRT-PCR, and data were normalized for mRNA levels of Hprt and compared by ANOVA using Bonferroni’s correction for multiple tests. Values are depicted as lower quartile, median, and upper quartile (boxes) with minimum and maximum ranges and statistically significant differences are indicated (n = 4 independent experiments). (B) Fpn1 and actin protein levels were assessed by Western blotting, with one of four independent experiments shown. (C) Fpn1 and actin levels were quantified by densitometric scanning of membranes (n = 8 independent experiments). (D–F) 59Fe transport studies were used to determine macrophage iron release and bacterial iron acquisition within macrophages and in liquid culture, respectively. (D) After loading with 10 µM 59Fe-citrate, macrophage iron release was determined (n = 6 independent experiments). (E) Bacterial iron acquisition within macrophages was determined after loading of infected macrophages with 10 µM 59Fe-citrate. Data were normalized for Salmonella numbers as determined by plate counting (n = 6 independent experiments). (F) Bacterial iron acquisition in liquid culture was determined after incubation with 10 µM 59Fe-citrate. As control, an isogenic triple mutant, deficient in enterobactin synthesis, sitABCD-, and feo-mediated iron uptake (S. tm. entC sit feo) was used. Data are depicted relative to the solvent-treated control (Ctrl.) as described in A (n = 4 independent experiments). (G) Wild-type PE-MΦ were infected with Salmonella typhimurium (S. tm.) and Nrf2-binding activity was determined by a commercially available assay and depicted as arbitrary units (AU; n = 8–10 independent experiments). (H) Wild-type PE-MΦ were transiently transfected with an 8.4-kb Fpn1 promoter luciferase construct (Fpn1 8.4-kb), a variant carrying a specific mutation in the Nrf2-binding site (Nrf2 mut.), or empty plasmid (not depicted), and subsequently treated with PBS or infected with S. typhimurium (S. tm.). Luciferase activity is expressed as arbitrary light units (ALU; n = 4 independent experiments). **, P < 0.01; ***, P < 0.001.

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