Fig 1: miR-155 is induced and MafB is suppressed by eCIRP in ischemic stroke. a,b miR-155 host gene MiR155hg (a) and Mafb (b) expression Z-scores in microglia cluster from GSE227651. c Relative levels of miR-155 in hippocampus 24 h after stereotactic injection of PBS or 1 μg eCIRP (Contra. & Ipsi. indicate hippocampus laterality relative to eCIRP injection). (n = 7–8; mean ± SEM; * p = 0.0009 vs. Sham & p = 0.0002 vs. Contra.; ns. p = 0.9632 vs. Sham; one-way ANOVA, Tukey’s multiple comparisons test). d Representative Western blot images and relative MafB protein levels in hippocampal tissues 24 h after stereotactic injection of PBS or 1 μg eCIRP (n = 4–6; mean ± SEM; * p = 0.0416 vs. Sham & p = 0.0159 vs. Contra.; ns. p = 0.946 vs. Sham; one-way ANOVA, Tukey’s multiple comparisons test). e Relative brain levels of miR-155 in WT and CIRP−/− mice subjected to sham and tMCAO surgery. (n = 5; * p = 0.0217 WT sham vs. WT tMCAO; ns. p = 0.903 CIRP−/− sham vs. CIRP−/− tMCAO; two-way ANOVA, Tukey’s multiple comparisons test). f Representative Western blot images and relative MafB protein levels normalized to β-actin in whole brain lysates of WT and CIRP−/− mice subjected to sham and tMCAO surgery. (n = 3–7; * p = 0.046 WT sham vs. WT tMCAO; ns. p = 0.7657CIRP−/− sham vs. CIRP−/− tMCAO; Mixed-effects model (REML) with Sidak’s multiple comparisons test).
Fig 2: eCIRP is released in ischemic stroke and causes efferocytic dysfunction in vivo. a Concentration of eCIRP in CSF collected from mice 24 h after sham or tMCAO surgery. (n = 5; mean ± SEM; * p = 0.0006 vs. Sham; Unpaired t-test). b Mertk expression Z-score in microglia cluster from GSE227651. c Bederson score in WT tMCAO and CIRP−/− tMCAO mice 24 h after surgery. (n = 8; mean ± SEM; * p = 0.0031; Unpaired t-test). d Representative confocal images of peri-infarct brain tissues in Sham, WT tMCAO, and CIRP−/− tMCAO mice, stained for DAPI (blue), Iba-1 (magenta), and MerTK (green). Magnification 630x, scale bar, 20 μM. e Quantification of MerTK expression in microglia per animal. (n = 6; mean ± SEM; * p < 0.0001 vs. Sham; # p = 0.0032 vs. WT tMCAO; one-way ANOVA, Tukey’s multiple comparisons test). f Representative confocal images of peri-infarct brain tissues in WT tMCAO and CIRP−/− tMCAO mice, with staining for nuclei (DAPI, blue), neurons (NeuN, yellow), dead/dying cells (TUNEL, green), and microglia (Iba-1, magenta); arrows indicate efferocytosis events with complete internalization of TUNEL+/NeuN+ cargo, arrowheads indicate incomplete efferocytosis events. Magnification 630x, scale bar, 20 μM. g Ratio of TUNEL+ neurons efferocytosed by microglia to total TUNEL+ neurons expressed as fold-change with WT tMCAO set as 1. (n = 8; mean ± SEM; * p = 0.0017 vs. WT tMCAO; Unpaired t-test). h Zoomed in high power reconstruction of 3D segmented surfaces in Imaris showing efferocytosis event indicated in white box in f, demonstrating TUNEL+/NeuN+ cargo engulfed by Iba-1+ microglia.
Supplier Page from Fine Biotech Co., Ltd. for Mouse Cirbp (Cold-inducible RNA-binding protein) ELISA Kit