Fig 1: Microglia accumulate lipid droplets after brain ischemia Expression of a selection of genes encoding lipid droplet-associated proteins in microglia sorted from control (n = 4) and ischemic (n = 4) male mice at day four postischemia (obtained from RNAseq data shown in Fig 1). Heatmap illustrates ischemia-induced upregulation of genes marked with * indicating adjusted P-value < 0.001.mRNA expression of PLIN2 and ISG15 in postmortem human brain tissue of nine stroke patients (points are values of individual patients); eight women (red points) and one man (black point). Samples were obtained from the ischemic tissue (Stroke) and nonaffected (NA) tissue. The graph shows boxes from the 25th to 75th percentiles, the median line, and whiskers down to the minimum and up to the maximum value, showing all points. Values are expressed as fold versus mean control (i.e., nonaffected tissue) Wilcoxon matched-pairs signed-rank test, **P = 0.0039; *P = 0.0195.PLIN2 protein expression in mouse brain tissue, as assessed by Western blotting. Values were obtained from the ipsilateral hemisphere 1 h (n = 3), 4 h (n = 3), 15 h (n = 4), 24 h (n = 5) and 96 h (n = 5) postischemia, and 15 h (n = 4), 24 h (n = 2), and 96 h (n = 2) after sham operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1 h, n = 2; 24 h, n = 3; and 96 h, n = 3) were also evaluated. Ischemia increased PLIN2 expression at day 4 versus the sham group (**P = 0.0032, Kruskal–Wallis test and Dunn's multiple comparisons test). ß-tubulin is the protein loading control. The “Std” lane indicates the molecular weight standard. Quantification of band intensity where values are expressed as fold versus control (nonischemic). Points correspond to independent male mice and group values are expressed as a violin plot. Values were obtained 1 h (n = 3), 4 h (n = 3), 15 h (n = 4), 24 h (n = 5), and 96 h (n = 5) postischemia, and 15 h (n = 4), 24 h (n = 2), and 96 h (n = 2) after sham operation. Values of sham mice were pooled together since they did not differ between time points. Samples of the contralateral hemisphere (Contra) of ischemic mice (1 h, n = 2; 24 h, n = 3; and 96 h, n = 3) were also evaluated. Ischemia increased PLIN2 expression at day 4 (**P = 0.0032, Kruskal–Wallis test and Dunn's multiple comparisons test versus the sham group). Data are presented as violin plots with lines at the median and quartiles (dashed lines).Immunofluorescence with antibodies against PLIN2 (green) and Iba-1 (red) in brain tissue 1 day after induction of ischemia in female mice. Nuclei are stained with DAPI (blue). Control is the contralateral hemisphere. Scale bar: 10 µm.Transmission electron microscopy showing microglial cells in control or ischemic tissue 1 day postischemia. Ischemia induces the formation of lipid droplets (LD) in microglia. LD are often seen near lysosomes (Lys). Insets in the lower panels are magnifications of the regions marked with a square. Scale bar 2 µm.Flow cytometry gates to identify Bodipy+ microglia for control and ischemic brain tissue. Gates were set based on fluorescence minus one (FMO) intensity values.Quantification of the percentage of CD11b+CD45low microglia containing lipid droplets as Bodipy+ microglia using flow cytometry in male mice deficient in Stat1 (Stat1-/-; n = 4) and corresponding Stat1+/+ mice (n = 7) shows ischemia-induced increases in Stat1+/+ mice (**P = 0.0086) but not in Stat1-/- mice (P = 0.927; Two-way ANOVA and Šídák's multiple comparisons test). The graph shows values of the nonischemic (-) and ischemic (+) brain hemispheres of individual mice and the mean ± SD.