Fig. 3

FOXO1 enhances the anti-apoptosis and IL-6 release abilities of neutrophils. a Flow cytometry showing the percentage of apoptosis in neutrophil infiltrate to brain injury in TBI mice (n = 5). b ELISA analysis (left) and flow cytometry (right) of IL-6 levels in neutrophils of mice collected after TBI (n = 5). c Western blotting showing the expression of VCAN in neutrophils in both TBI patients and TBI mouse models. Three biologically independent samples of each group represented the average status here. d Flow cytometry showing the percentage of apoptosis in neutrophils transfected with different over/si-expression plasmids. Three biologically independent samples to represent the average status. e ELISA analysis of IL-6 in neutrophils of mice. The experiment was repeated three times. f Putative FOXO1 binding sequence of the mouse VCAN promoter gene. VCAN mut 1: deletion of one FOXO1 binding site at − 1874 to − 1885 bp; VCAN mut 2: deletion of two FOXO1 binding sites at − 1874 to − 1885 bp and − 1281 to − 1292 bp; VCAN mut 3: deletion of three FOXO1 binding sites at − 1874 to − 1885 bp, − 1281 to − 1292 bp and − 802 to − 813 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 VCAN 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 The RMSD showed by the backbone atoms of the VCAN-BAX system during the molecular docking. j Pymol MOE software was used to predict the binding between VCAN and BAX. k Co-IP of VCAN and BAX in neutrophils from bone marrow in a mouse model. β-actin was detected in the supernatant after IP. l Immunostaining showing the expression, location, and binding between BAX and VCAN in neutrophils from bone marrow in the mouse model of TBI. Scale bar = 1 μm. m Immunostaining showing the expression of the neuron marker NeuN (green) and nuclei (DAPI, blue) in neurons co-cultured with neutrophils treated as described above. Scale bar = 50 μm. The experiment was repeated 5 times. Data are represented as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns non-significant. FOXO1 forkhead box protein O1, TBI traumatic brain injury, ELISA enzyme‐linked immunosorbent assay, ORF open reading frame, mut mutant, ChIP chromatin immunoprecipitation, VCAN Versican, RMSD root mean square deviation, BAX BCL-2-associated X protein, DAPI 4,6-diamidino-2-phenylindole dihydrochloride, Co-IP co-immunoprecipitation