Fig 1: ILF3 is necessary for translation of ISGs. (A) Heatmap of ISGs enrichment in polysomal fractions (n = 374) during homeostasis (mock), versus activated type I IFN response (siMock p(I:C)) and activated IFN response in the absence of ILF3 (siILF3 p(I:C)). (B) Heatmap of TOP mRNAs enrichment in polysomal fractions (n = 45) in the same conditions as in (A) (C) qRT-PCR quantification of ISG enrichment in subpolysomal and polysomal pooled fractions in mock (-p(I:C)) or stimulated (+p(I:C)) HeLa cells, in the presence or absence of ILF3. Data show the average (n = 3) ± s.e.m, normalized to 18S rRNA and relative to subpolysomal level in mock (*) P l< 0.05, (**) P l< 0.001 by two-way ANOVA followed by Tukey's multiple comparison test, N.S, non-significant. (D) IFIT3, OASL and IRF1 western blot analyses upon dsRNA stimulation (lane 2), and in the absence of ILF3 (lane 4), tubulin and fibrillarin serve as loading controls.
Fig 2: Proliferative microglia cluster emerged in the ischemic mice brain. A t-SNE representation of MG4 (left); violin plots showing the top up-regulated genes in MG4 (right). B Feature plots displaying cell cycle score determined by expression of highly conserved cell cycle genes. C, D Representative confocal images of MKI67 and IBA-1 double immunostaining in the ischemic penumbra and quantification of the MKI67+/IBA-1+ cells at different timepoints after tMCAO. Scale bar: 40 µm (overviews). Triangles indicate resident MKI67+ microglia. one-way ANOVA with Bonferroni multiple comparisons test. n = 6–8. The data are shown as means ± SD. *p < 0.05, ***p < 0.001, n.s. no significance. E, F Representative confocal images of CH25H/OASL, IBA-1 and EdU treble immunostaining in the ischemic penumbra 3d after tMCAO. (tMCAO transient middle cerebral artery occlusion, t-SNE t-distributed stochastic neighbor embedding, MG microglia, IBA-1 ionized calcium binding adapter molecule 1, ANOVA analysis of variance.)
Fig 3: OASL+ microglia subset exhibits type I interferon upregulated signaling and was associated with increased infarct volume after stroke. A t-SNE representation of MG5 (left); violin plots showing the top up-regulated genes in MG5 (right). B Enriched GO terms in MG5. C Top predicted upstream transcriptional regulators of MG5 activation. D Schematic representation of the experimental design. E Cerebral blood flow was monitored using two-dimensional laser speckle imaging techniques on the left side before, during, and 3 days after distal middle cerebral artery occlusion (dMCAO). The values on the right side were expressed as a percentage change from the baseline measurements taken prior to dMCAO. F Quantification of infarct volume 3 days after dMCAO of young (3 months) and aged (21 months) mice. n = 6 per group. G Representative images of microglia in adult brain and aged brain after ischemic stroke. Scale bars, 30 µm. H OASL+ quantified in microglia (IBA1+) in the brain of young (3 months) and aged (21 months) mice of sham and ischemic stroke mice (young, n = 12; aged, n = 12; two-sided t test; mean ± SD). I Correlation between OASL+ microglia proportion and infarct volume of tMCAO and dMCAO. Data are presented as means ± SD. (t-SNE t-distributed stochastic neighbor embedding, MG microglia, GO gene ontology, DEG differentially expressed genes, IBA-1 ionized calcium binding adapter molecule 1, OASL 2'–5'-oligoadenylate synthetase-like, tMCAO transient middle cerebral artery occlusion, dMCAO distal middle cerebral artery occlusion; ANOVA analysis of variance)
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