Fig. 4
Assessment of ROS generation in SCV in the presence or absence of FPN. a Protein carbonylation in SCV of Raw264.7 cells treated with PBS, hepcidin, or DFO, as determined using 2,4-dinitrophenylhydrazine. SCV were isolated using magnetized Salmonella (MOI, 100) 2 h p.i. Proteins with 2,4-dinitrophenylhydrazine groups were quantified using a specific antibody. b Lipid peroxidation was determined in cells treated as in a. c–g ROS biosensor Salmonella (katGp-gfpOVA16) that fluoresce green in the presence of hydrogen peroxide were used to assess ROS levels in SCV. c Green fluorescence of katGp-gfpOVA-expressing Salmonella treated with 10 μM H2O2. d Expression of katGp-gfpOVA in the Salmonella infecting Raw264.7 cells pretreated with PBS, hepcidin (1 μg/ml), or DFO (100 μM). Images were taken 2 h p.i. using a confocal microscope. f Expression of katGp-gfpOVA in the Salmonella infecting C57BL/6 mice treated with GSK5182 (FPN+) or PBS (FPN−). Spleens of mice infected with Salmonella i.v. (1 × 105 CFU) were examined by confocal microscopy 2.5 days p.i. d, f Nuclei were stained with DAPI (blue), Salmonella with a specific antibody (red), and katGp-gfpOVA expression is shown in green. Scale bars, 10 μm. e, g The fractions of biosensor-positive (green) cells were quantified as a percentage of the total intra-macrophage Salmonella (red) in the experiment shown in d, f. Data are presented as means ± SEM. Significance is indicated as ****p < 0.0001 by two-tailed Student’s t-test
