Extended Data Fig. 5: Deletion of Itgb3 in CSF1R⁺ cells exacerbates atherosclerosis and induces proliferation and accumulation of this cell lineage in the plaque.

a–j, Apoe^(−/−), Csf1r-Mer-iCre-Mer mice also carrying Itgb3^(fl°^x/flox) or wild type for Itgb3 were induced with tamoxifen. In a–f, mice were then fed a WD for 16 weeks, and transverse aortic root sections were stained with H&E (dashed lines demarcate lesion and necrotic core in a,e, respectively) and Oil Red O (b). Quantification of lesion area (c), lipid content (d) and necrotic core area (f). n = 5 mice and 10–12 plaques per genotype (triplicate measurements per plaque). BM-derived GFP⁺ myeloid cells (Ly6C⁺) were isolated by FACS after 5 days of tamoxifen induction and subjected to qRT-PCR for Itgb3 (g). Itgb3 mRNA is relative to Gapdh and normalized to control. n = 6 mice per group, and qRT-PCR was done in triplicate. Four weeks following tamoxifen induction, peripheral blood total cholesterol and triglycerides (after a 16 h fast; n = 5–7 mice per group; h,i) and leukocyte counts (j) were determined. n = 5 and 7 mice in Itgb3^(+/+) and Itgb3^(flox/flox) groups, respectively. k–o, Apoe^(−/−), Csf1r-Mer-iCre-Mer, ROSA26R^(mTmG/+) mice also carrying Itgb3^(+/+) or Itgb3^(flox/flox) were induced with tamoxifen and fed a WD for 16 weeks. In k,l, after 16 weeks of WD feeding, EdU was injected intraperitoneally 12 h prior to euthanasia. Transverse aortic root sections were stained for markers of fate (GFP), SMCs (SMA), nuclei (DAPI) and either proliferation (EdU) in k or CD68 in m. Boxed regions in k are shown as close-ups below with arrowheads indicating GFP⁺EdU⁺ cells. In l, the percentage of GFP⁺ plaque cells that are EdU⁺ is shown. In, n,o, percentage of plaque cells that are GFP⁺ or CD68⁺ was quantified. n = 5 and 7 mice in Itgb3^(+/+) and Itgb3^(flox/flox) groups, respectively, 10 plaques per genotype, 4 sections with a total of ~1000–1250 cells and spanning 150 μm per plaque (l,n,o). All data are means ± SD, and two-tailed Student’s t-test was used. Lu, lumen; Med, media; Pl, plaque. Scale bars, 100 μm (a,b,e), 50 μm (k,m).