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. Author manuscript; available in PMC: 2023 Aug 11.
Published in final edited form as: Br J Pharmacol. 2022 Aug 3;179(21):4974–4991. doi: 10.1111/bph.15926

FIGURE 7.

FIGURE 7

Homozygous deletion of Pfkfb3 in myeloid cell exacerbates atherosclerosis. (a, b) Oil red O-stained en face aortic preparations from 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (a), and quantification of the oil red O-stained areas (b) (n = 12 mice per group). (c, d) Oil red O staining of aortic roots (c), and quantification of lesion areas (d) in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 7 mice per group). (e, f) IHC staining of Mac2 (e), and quantification of Mac2+ areas (f) in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 6 mice per group). (g, h) H&E staining of aortic roots (g), and quantification of necrotic areas (h) in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 7 mice per group). Red lines show the boundaries of necrotic cores. (i, j) cleaved caspase 3 staining (i) and quantification of apoptotic bodies (j) in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 7 mice per group). (k, l) representative flow cytometry phagocytosis plots (l), and quantificative analysis of efferocytosis (k). Peritoneal macrophages from Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice were incubated for 45 min with apoptotic Jurkat T cells, and then were assessed for efferocytosis by flow cytometry (n = 6). (m) Representative immunofluorescent staining of cleaved caspase 3 (red) and Mac2 (green) in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 6 mice per group). (n) the number of “free” apoptotic bodies not associated with Mac2+ phagocytic macrophages in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 6 mice per group). (o) The number of caspase 3 and Mac2 double-positive macrophages in aortic roots of 3-month-WD-fed Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− mice (n = 6 mice per group). (p) F-actin formation during efferocytosis in Apoe−/−/Mye-Pfkfb3+/+ and Apoe−/−/Mye-Pfkfb3−/− macrophages. Peritoneal macrophages were co-incubated with CFSE-labelled apoptotic Jurkat T cells for 30 min and stained with phalloidin. Actin polymerization was measured by flow cytometry (n = 5). MFI, mean fluorescence intensity; rel., relative; MΦ, macrophage. For all bar graphs, data are mean ± SEM, *P < 0.05 (unpaired, two-tailed Student’s t-test for a–o; one-way ANOVA with Tukey’s post hoc test for p).