Fig. 2

Changes in protein expression levels and cellular localization of iron-related ferroptosis markers. A Western blot data showing increased expression of TfR1 (onset), DMT1 (onset and peak), HO-1, ferritin, and NCOA4 (peak and progressive stages) of CH-EAE. (Ai:TfR1; naïve vs. onset and prog: p = 0.04 and p = 0.005 respectively; F_{(3, 16)} =  17.21, p < 0.001, Aii: DMT1; naïve vs. onset and peak: p = 0.03 and p < 0.001 respectively; F_{(3, 15)} =  57.40, p < 0.001, Aiii: HO-1; naïve vs. peak and prog: p = 0.02 and p = 0.03 respectively; F_{(3, 16)} = 7.956, p = 0.002, Aiv: ferritin; naïve vs. peak and prog: p = 0.04 and p < 0.001 respectively; F_{(3, 16)} = 31.42, p < 0.001, Av: NCOA4; naïve vs. peak and prog: p < 0.001 respectively; F_{(3, 15)} = 31.10, p < 0.001. n = 4–5 per group, one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001). B–F Double immunofluorescence labeling with rabbit anti-ferritin/rat anti-CD11b (B), rabbit anti-ferritin/mouse anti-CC1 (C), rabbit anti-HO-1/rat anti-CD11b (D), rabbit anti-NCOA4/rat anti-CD11b (E), and rabbit anti-NCOA4/mouse anti-CC1 (F). Areas in white rectangles are shown at higher magnification at the bottom of each panel. B Shows localization of ferritin in CD11b⁺ macrophages (53.1 ± 4.0%) and C in CC1⁺ oligodendrocytes (37.6 ± 3.9%) in the progressive stage of CH-EAE. D Shows localization of HO-1 in CD11b⁺ macrophages at the peak stage of CH-EAE (41.4 ± 2.68%). E Shows localization of NCOA4 in CD11b⁺ macrophages (49.6 ± 4.4%) (E), and F in CC1⁺ oligodendrocytes (42.8 ± 3.5%) F in the progressive stage of CH-EAE. Scale bars: 40 µm; Inset:12 µm