Fig. 4

Changes in expression levels and cellular localization of ferroptosis markers related to lipid repair and GSH metabolism. A Western blot analysis of ACSL4 (Ai) and LPCAT3 (Aii) show increased expression in later stages of CH-EAE. (Ai ACSL4, naïve vs. peak and prog: p = 0.03 and 0.04 respectively; F_{(3, 16)} = 5.81, p = 0.007; Aii LPCAT3, naïve vs. prog: p = 0.04; F_{(3, 16)} = 3.755, p = 0.03). Aiii The percentage of CC1⁺ oligodendrocytes expressing ACSL4 increases significantly from naïve to the peak and progressive stages of CH-EAE (naïve vs. peak and prog: p < 0.001; F_(3,16) = 42.65, p < 0.001) B Double immunofluorescence labeling shows that ACSL4 is expressed in CC1⁺ oligodendrocytes. Expression in oligodendrocytes is increased in the peak and progressive stages of CH-EAE. C Western blot analysis of xCT (Ci) and GPX4 (Cii) show reduced expression in the peak and progressive stages of CH-EAE (Ci:  xCT; naïve vs. peak and prog: p = 0.009 and p = 0.04 respectively; F_{(3, 16)} = 6.952, p = 0.003, Cii:  GPX4; naïve vs. peak and prog: p < 0.001; F_{(3, 16)} = 20.67, p < 0.001) Panel Ciii quantification showing a gradual reduction in the percentage of CC1⁺ oligodendrocytes expressing GPX4 (naïve vs. onset, peak and prog: p = 0.02 and p < 0.001; F_{(3, 12)} = 17.32, p < 0.001) Ai-iii and Ci-iii: n = 4–5 per group, one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001. Panel Civ shows reduction in GSH levels at all stages of CH-EAE. (Naïve vs. onset, peak and prog: p = 0.01 and p < 0.001 respectively; F_{(3, 20)} = 11.90, p < 0.001, n = 6 per group, one-way ANOVA with Tukey’s multiple comparison test, **p < 0.01 and ***p < 0.001). D Double immunofluorescence co-localization of GPX4 in CC1⁺ oligodendrocytes. Scale bars: 40 µm; Inset: 12 µm