Fig. 3
Assessment of SCV iron content in the presence or absence of FPN. a Expression of the iron biosensor in the presence or absence of iron chelator (2,2′-bypyridyl, 0.2 mM) in vitro. All Salmonella were stained with DAPI (blue), and iroBp-gfpOVA-expressing Salmonella are green. b Salmonella growth was determined in RPMI 1640 supplemented with the indicated concentration of FeSO4 (0.005–500 μM). Bacterial cell mass (A600) was measured at the indicated times. c Expression of iroBp-gfpOVA at 2 h p.i. in Salmonella grown as in b was quantified as the percentage of GFP+ Salmonella, as shown in a. Data are presented as means ± SEM. Significance is indicated as ****p < 0.0001 by two-tailed Student’s t-test. d, f Nuclei were stained with DAPI (blue), Salmonella with a specific antibody (red), and iroBp-gfpOVA expression is shown in green. d Expression of iroB-gfpOVA was determined in the Salmonella (MOI, 100) infecting Raw264.7 cells pretreated with PBS, hepcidin (1 μg/ml), FeSO4 (0.1 mM), or DFO (100 μM). Images were taken 2 h p.i. by confocal microscopy. f Expression of iroB-gfpOVA in the Salmonella infecting C57BL/6 mice treated with GSK5182 (FPN+) or PBS (FPN-). Representative images of spleens infected with Salmonella were examined by confocal microscopy 2.5 days after i.v. infection (1 × 105 CFU). Scale bars 10 μm. e, g The fraction of iroBp-gfpOVA-expressing (green) Salmonella shown in d, f was quantified, respectively, as in b. Data are presented as means ± SEM. Significance is indicated as ***p = 0.0004; ****p < 0.0001 by two-tailed Student’s t-test
