Fig 2: Distinguishing indicators and regulators of ferroptosis in different brain regions of ECM mice. a and b MDA levels (a) and Fe contents (b) in the cerebrum and brainstem of control and ECM mice were measured using a commercial kit. c Expression of TfR1, ACLS4, and GPX4 in the olfactory bulb, cerebrum, cerebellum, and brainstem of control and ECM mice was measured by western blotting. Relative quantitative analysis was performed densitometry. d mRNA expression of tfrc, acsl4, and gpx4 in the olfactory bulb, cerebrum, cerebellum, and brainstem of control and ECM mice was measured by quantitative real-time polymerase chain reaction. Relative expression is shown as fold-change in relation to control levels. Data are reported as mean ± standard deviation. Each point represents one animal. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. ACSL4 acyl-CoA synthetase long-chain family member 4, ECM experimental cerebral malaria, GPX4 glutathione peroxidase 4, MDA malondialdehyde, TfR1 transferrin receptor 1

Fig 3: CD8+ T cells infiltrate the cerebrum of ECM mice and induce ferroptotic damage to primary neurons. a CD8+ T cells infiltrating the cerebrum of ECM mice were co-labeled with CD3 (green) and CD8 (red), and detected by immunofluorescence staining. The nucleus was stained with DAPI (blue). b Morphological changes in primary neurons were observed by microscopy (200 × magnification). c Expression of APP and MAP2 in primary neurons, as measured by western blotting, after co-culture with naïve or activated CD8+ T cells. d Expression of APP and MAP2 in primary neurons, as measured by western blotting after co-culture with activated CD8+ T cells with or without Fer-1. e Relative mitochondrial membrane potential (MMP) levels were calculated based on the optical density value and shown as the fold change relative to the blank value. Data are reported as mean ± standard deviation. Each point represents one sample. *p < 0.05 and ***p < 0.001 vs. control or activated CD8+ T cells. f Expression of TfR1 and GPX4 in primary neurons, measured by western blotting after co-culture with activated CD8+ T cells for 2–24 h. APP amyloid-beta precursor protein, ECM experimental cerebral malaria, GPX4 glutathione peroxidase 4, MAP2 microtubule-associated protein 2, TfR1 transferrin receptor 1