Figure 7
From: H-Ferritin is essential for macrophages’ capacity to store or detoxify exogenously added iron
The absence of H-ferritin is associated with higher levels of iron-induced oxidative stress. (a,b) Fth1+/+ (black) and Fth1−/− (grey) macrophages were treated with increasing concentrations of (a) FAC or (b) Hemin, in the absence (solid lines) or presence (dashed lines) of NAC, for 3 days. Cell death was measured through SYTOXTM Green incorporation into dead cells. The results represent the mean ± SD of four to seven independent cultures per condition (two-way ANOVA with Tukey multiple comparisons test; *p < 0.05). (c,d) Fth1+/+ (black) and Fth1−/− (grey) BMDM were treated with increasing concentrations of (c) hydrogen peroxide or (d) tBHP, for 15 minutes. Cell death was analyzed through SYTOX Green incorporation into dead cells. The results represent the mean ± SD of three independent cultures per condition. (e,f) Fth1+/+ (black) and Fth1−/− (grey) BMDM were kept untreated (circles) or treated with 10 µM FAC (square) or 100 µM hemin (triangle). (e) ROS production was analyzed through oxidation of DCFDA dye. The results represent the mean ± SD of three independent cultures per condition and are expressed as the percentage of macrophages marked with the fluorescent-oxidized dye. (f) Lipid peroxidation was assessed through BODIPY assay. The results represent the mean ± SD of three independent cultures per condition and are expressed as the ratio between peroxidized lipids (intracellular fluorescence intensity at 510 nm) relative to the total lipids (intracellular fluorescence intensity at 510 nm plus at 590 nm); (two-way ANOVA with Tukey multiple comparisons test; *p < 0.05). (g,h) Fth1+/+ (black) and Fth1−/− (grey) BMDM were treated with 10 µM FAC or 100 µM hemin for 24 hours. (g) Detection of carbonylated proteins by slot blot. The image corresponds to one representative experiment with BMDM obtained from 1 animal per genotype. Cell extracts were blotted in duplicate. The whole membrane is shown. (h) Densitometry analysis with ImageLabTM software of carbonylated proteins. The bars represent the mean + SD from 4 independent cultures, of the relative density of the bands obtained from Fth1+/+ (black) or Fth1 −/− (grey) BMDM in each condition, as compared to non-treated Fth1+/+ cells.
