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Fig. 6 | Journal of Neuroinflammation

Fig. 6

From: All trans-retinoic acid protects against acute ischemic stroke by modulating neutrophil functions through STAT1 signaling

Fig. 6

All-trans retinoic acid modified neutrophil functions through STAT1 signaling. a, b Expression of RAR-α, -β, and -γ in neutrophil was illustrated with immunofluorescent staining (a) and Western blot (b). c Heat map showing the modification of atRA treatment to STATs signaling in neutrophil. In the heat map, data are displayed as fold change to neutrophils from DMSO-treated group. Black, expression un-altered, fold change = 1; Red, expression up-regulated, fold change > 1; Green, expression downregulated, fold change < 1. d Quantification of the mRNA expression of STAT1 in neutrophil after atRA treatment. Quantification of markers that without significant difference between the two groups were displayed in Additional file 1: Figure S3. Experiments were repeated for 3 times. ***P ≤ 0.001, versus DMSO treated group in t test. e Protein expression of STAT1, phosphorylated STAT1 (pSTAT1, Y701), and SOCS1 in neutrophil after atRA treatment. Experiments were repeated for three times. *P ≤ 0.05, versus DMSO-treated group in t test. f Representative image of ChIP experiments showing that RARs could bind to SOCS1 gene. Experiments were repeated for three times. g Anisomycin (25 μg/ml) was treated to neutrophil to over-activate STAT1 signaling and abrogate the suppression of STAT1 phosphorylation by SOCS1. Neutrophil expression of N2 markers CD206 and IL-10 was assessed with flow cytometry. Experiments were repeated for three times. **P ≤ 0.01, ***P ≤ 0.001 in one-way ANOVA. hj Activation status of STAT1 signaling in neutrophil in the ischemic brain was assessed with flow cytometry at 1–3 days after tMCAO. Neutrophil in the stroke lesion of atRA pre-treated mice displayed down-regulated STAT1 phosphorylation (h, starting at 2 days), upregulated SOCS1 expression (i, starting at 1 day), and decreased IFNγ expression (j, starting at 2 days). Representative flow panels of the expression of pSTAT1, SOCS1, and IFNγ were shown. Percentage among CD11b+CD45hiLy6G+ neutrophil were calculated and displayed. N = 4 mice per group. *P ≤ 0.05, ***P ≤ 0.001, versus PBS-treated group in two-way ANOVA

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