Fig 1: Schematic diagram illustrated the KDM6B/IRF4/Notch2/SOX9 axis in ischemic brain injuryDemethylase KDM6B promotes the expression of IRF4 through the demethylation of the IRF4 promoter. IRF4 promotes the expression of Notch2 and its downstream gene SOX9 by binding to the Notch2 promoter, which in turn aggravates ischemic brain injury.
Fig 2: The expression of Notch2 and its downstream gene SOX9 was elevated by IRF4 via binding to the promoter region of Notch2(A) The KEGG enrichment analysis of 1,087 genes obtained from the bioinformatics online tool DAVID; the abscissa represents GeneRatio, and the ordinate represents the pathway ID and name. (B) The binding sites of IRF4 and Notch2 in the upstream and downstream regions obtained from the Chipbase v.2.0 website. (C) The co-expression relationship between IRF4 and Notch2. (D) The co-expression relationship between IRF4 and SOX9. (E) Luciferase activity of Notch2 promoter or promoter mutant plasmids in 293T cells after co-transfection with IRF4. (F) The binding of IRF4 to the Notch2 promoter region with or without OGD/R treatment determined by ChIP assay. (G) IRF4 knockdown efficiency in astrocytes determined by western blot analysis. (H) The mRNA expression of IRF4, Notch2, and SOX9 after OGD/R treatment determined by qRT-PCR. (I) The protein expression of IRF4, Notch2, and SOX9 after OGD/R treatment determined by western blot analysis. (J) The mRNA levels of IRF4, Notch2, and SOX9 after OGD/R treatment determined by qRT-PCR. (K) The protein levels of IRF4, Notch2, and SOX9 after OGD/R treatment determined by western blot analysis. Data in the figure were all measurement data and expressed by mean ± standard error of mean. Comparison between two groups was analyzed by unpaired t test, and comparison among multiple groups was analyzed by one-way ANOVA with Tukey’s test. The experiment was repeated three times. **p < 0.05.
Fig 3: Demethylase KDM6B deteriorates ischemic brain injury in mice via the IRF4/Notch2/SOX9 axis(A) The neurological deficit score of MCAO-operated mice. (B) The behaviors of MCAO-operated mice evaluated by pole test and foot fault test. (C) The cerebral infarction area in MCAO-operated mice shown by TTC staining. (D) Astrocyte activation determined by GFAP immunofluorescence staining. (E) Neuronal apoptosis determined by TUNEL staining. (F) The expression of inflammatory factors TNF-a, IL-1ß, and IL-6 in mouse serum determined by ELISA. (G) The KDM6B, IRF4, Notch2, and SOX9 mRNA expression in the brain tissue of mice determined by qRT-PCR. (H) The KDM6B, IRF4, Notch2, and SOX9 protein expression in the brain tissues of mice measured by western blot analysis. Data in the figure were measurement data and expressed by mean ± standard error of mean. Comparison between two groups was analyzed by unpaired t test, and comparison among multiple groups was analyzed by one-way ANOVA with Tukey’s test. The experiment was repeated three times. **p < 0.05.
Fig 4: GSEA enrichment plot of KEGG Notch signaling pathway genes and MAP4K4 RNAi decreased gene expression. (A) Genes in the KEGG Notch signaling pathway revealed a significant enrichment in patients with a higher expression of MAP4K4 compared with patients with lower expression. The top portion of the figure plots the enrichment scores for each gene and the bottom portion of the plot shows the value of the ranking, moving down the list of ranked genes. (B) Reverse transcription-quantitative polymerase chain reaction and (C) western blot analysis revealed that the expression levels of Notch2, Notch3 and Hes1 were significantly reduced by MAP4K4 RNAi. The data are expressed as the mean ± standard deviation (**P<0.01). KEGG, Kyoto Encyclopedia of Genes and Genomes; RNAi, RNA interference; WT, wild-type; Neg., negative control; MAP4K4-Ri-3, MAP4K4-shRNA-3 virus-infected cells.
Fig 5: KDM6B promotes astrocyte inflammation by positively regulating the IRF4/Notch2/SOX9 axis(A) The mRNA expression of KDM6B, IRF4, Notch2, and SOX9 in OGD/R-exposed astrocytes determined by qRT-PCR. (B) The protein expression of KDM6B, IRF4, Notch2, and SOX9 OGD/R-exposed astrocytes determined by western blot analysis. (C) The apoptosis of OGD/R-exposed astrocytes detected by TUNEL assay. (D) The expression of inflammation factors TNF-a, IL-1ß, and IL-6 in OGD/R-exposed astrocytes determined by ELISA. Data in the figure were measurement data and expressed by mean ± standard error of mean. Comparison between two groups was analyzed by unpaired t test, and comparison among multiple groups was analyzed by one-way ANOVA with Tukey’s test. The experiment was repeated three times. **p < 0.05.
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