Fig. 6

FOXO1 disrupts iron homeostasis in neutrophils and oligodendrocytes. a Volcano plot showing variation in protein expression in brain-insured tissues between the group of TBI with depression and the group of TBI without depression in WT mice. The fold change (FC) log (base 2) is on the X-axis, and the negative false log discovery rate (P-value) (base 10) is on the Y-axis. Higher expression levels are indicated by red and lower by green. b Volcano plot showing variation in protein expression of neutrophils between the TBI-induced depression group and the insusceptible group. The FC log (base 2) is on the X-axis, and the negative false log discovery rate (P-value) (base 10) is on the Y-axis. Higher expression levels are indicated by red and lower by green. c Western blotting showing the expression of FOXO1, FTL11, TFRC, and xCT in neutrophils from the TBI mice with and without depression (n = 6). The sham group served as the control. In each group, three biologically independent samples were represented here to show the average status. d Intracellular iron colorimetric assay showing the concentration of total Fe in neutrophils from bone marrow of the TBI mice with and without depression (n = 5). The sham group served as the control. e LPO assay showing the concentration of LPO in neutrophils from the bone marrow of the TBI mice with and without depression (n = 5). The sham group served as the control. f Putative FOXO1 binding sequence of the mouse TFRC promoter gene. TFRC wt1: the reporter plasmid containing the two FOXO1 binding sites at − 1070 to − 1080 bp and − 1085 to − 1095 bp; TFRC mut1: the reporter plasmid containing the two mutated FOXO1 binding sites at − 1070 to − 1080 bp and − 1085 to − 1095 bp; TFRC wt2: the reporter plasmid containing the FOXO1 binding site at − 526 to − 536 bp; TFRC mut2: the reporter plasmid containing the mutated FOXO1 binding site at − 526 to − 536 bp. g Luciferase activity of FOXO1 co-transfected with mutated reporters under specific conditions. All transfected cells were treated under the indicated conditions for 6 h and lysed for dual-luciferase measurements. h ChIP of the FOXO1 binding sequence from the murine TFRC promoter gene. After treating neutrophils with the indicated conditions for 6 h, the total chromatin was collected and amplified as input (positive control). Antibodies against FOXO1 were used to pull down the binding segments, of which IgG was introduced as a negative control. i Electron microscopy image of the form of mitochondria in neutrophils. j Intracellular iron colorimetric assay showing the concentration of total Fe in neutrophils. k LPO assay showing the concentration of LPO in neutrophils from bone marrow. l Immunostaining of MBP (green) and nuclei (DAPI, blue) in oligodendrocytes (scale bar = 50 μm). Each experiment was repeated three times. Data are represented as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. FOXO1 forkhead box protein O1, TBI traumatic brain injury, wt wild-type, FTH1 ferritin heavy chain 1, FTL1 ferritin heavy chain 1, TFRC transferrin receptor, xCT system xc(−) cystine/glutamate antiporter, mut mutant, LPO lipid hydroperoxide, ChIP chromatin immunoprecipitation, MBP myelin basic protein, DAPI 4,6-diamidino-2-phenylindole dihydrochloride