Role of CB1 and oestrogen signalling in macrophage proliferation. (A) Representative images of proliferating (Ki67+, green and marked with white arrowheads) macrophages (CD68+, red) in aortic root plaques after 4 weeks Western diet (WD); nuclei were counterstained with Hoechst 33342 (blue). Scale bar: 100 μm (left) and 10 µm (right). (B) Total and relative counts of proliferating plaque macrophages (n = 4–6). (C, D) Flow cytometric analysis of proliferation rates in untreated male and female BMDMs isolated from Apoe^−/−LysM^Cre and Apoe^−/−LysM^CreCnr1^flox/flox mice (n = 5–9). (E) Proliferation rates in male BMDM treated with vehicle or 10 nM oestradiol (E2) for 24 h (n = 5–6). (F) Proliferation rates in female BMDM treated with vehicle or 1 µM tamoxifen (tam) for 24 h (n = 3–4). (G–J) Apoe^−/−LysM^Cre and Apoe^−/−LysM^CreCnr1^flox/flox female mice were fed with Western diet (WD) for 4 weeks and subjected to daily ip injections of 1 mg tamoxifen or vehicle for the last 3 days prior to analysis of proliferation rates in peritoneal macrophages (H; n = 4–7). (I) Representative immunostainings of proliferating (Ki67+) macrophages (CD68+) in aortic root plaques (nuclei, Hoechst); scale bar: 100 μm (left) and 10 µm (right). (J) Total and relative counts of proliferating plaque macrophages (n = 4). Each dot represents one mouse and all data are expressed as mean ± S.E.M. Two-sided unpaired Student’s t-test (J), two-way ANOVA followed by Tukey test (B, D–F, and H).