Fig 2: Identification of Lyve-1+ macrophages in the mammary gland.(A) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+ and CD45– Lyve-1+ cells by flow cytometry. (B) Mammary glands from 10-week-old mice (n = 4) were assessed for CD45+CD11b+F4/80+Lyve-1– and CD45+CD11b+F4/80+Lyve-1+ cells by flow cytometry. (C) Mammary glands were harvested from 6-week-old mice, immunostaining was performed for F4/80 and Lyve-1, and the localization of single- and double-positive cells associated with TEBs was examined (n = 5, three images/localization). Representative images of F4/80+Lyve-1– (i, arrowhead) and F4/80+Lyve-1+ (ii, arrowhead) cells are shown. Yellow lines show the margin of the mammary gland. Insets show higher magnification. (D, E) Representative images of F4/80+Lyve-1+ cells in the adipose stroma and fibrous capsule of the mammary gland. Representative images show F4/80 (iii) and co-staining (iv). Inserts show higher magnification. (F) Human mammary glands obtained from reduction mammoplasty samples demonstrate the presence of CD68+Lyve-1+ macrophages in the interlobular stroma (n = 5, three images/sample). Representative images show F4/80 (v) and co-staining (vi).Figure 1—source data 1.Source data for graphs in panels A and B.

Fig 3: PFKFB3 expression is increased in advanced atherosclerotic plaques and correlates with CD68+ macrophages and necrotic core area. (A,B) Increased expression of PFKFB3 (green) in atheromatous plaques compared with fibrous carotid plaques. n = 10 for fibrous and fibroatheromatous plaques, n = 8 for atheromatous plaques; one-way ANOVA, P = 0.0078 for fibrous vs. atheromatous plaques. Scale bar 50 µm. (A,C) CD68+ macrophages (red) increased in atheromatous carotid plaques. n = 10 for fibrous and fibroatheromatous plaques, n = 8 for atheromatous plaques; one-way ANOVA, P = 0.0008 for fibrous vs. atheromatous plaques. Scale bar 50 µm (D) PFKFB3 expression and CD68 expression correlated significantly. n = 28; linear regression analysis, R2 = 0.7456, P < 0.0001. (E) Human coronary atherosclerotic advanced plaques showed increased PFKFB3 expression compared to initial plaques. n = 4 for initial vs. n = 5 for advanced plaques; two-tailed unpaired Mann-Whitney, P = 0.0317. Scale bar 200 µm. (F) PFKFB3 expression positively correlated with necrotic core area. n = 9; Linear regression analysis R2 = 0.4861, P = 0.0368. (G) Immunohistochemistry of PFKFB3+ co-staining with CD68+ macrophages; Scale bar 100 µm, (H) PECAM+ endothelial cells; Scale bar 100 µm, (I) CD3+ T cells; Scale bar 100 µm, and (J) and a smooth muscle actin (aSMA)+ vascular smooth muscle cells (K); Scale bar 100 µm. Data are shown as mean ± standard error of the mean. *P < 0.05, **P = 0.005, ***P < 0.0005. PFKFB3, 6-phophofructo-2-kinase/fructose-2,6-biphosphatase 3; PECAM, platelet endothelial cell adhesion molecule; aSMA.

Fig 4: Immunohistochemistry contrasts ectopic and orthotopic RPE in age-related macular degeneration. This retina of a 90-year-old white female donor has huge soft drusen and continuous basal laminar deposit (BLamD) under the fovea. Yellow arrowheads, Bruch's membrane. Scale bars apply to each row. (A) RPE65 immunoreactivity of ectopic RPE including sloughed/intraretinal phenotypes presenting as hyper-reflective foci (HRF) at green/pink (RPE plume) arrowheads and shedding RPE phenotype at cyan arrowhead. (B) CD68 immunoreactivity of ectopic RPE including sloughed/intraretinal phenotypes at green/pink (RPE plume) arrowheads and shedding RPE phenotype at cyan arrowhead in the same retina in panel A. (C) Magnified view of ectopic RPE lacking RPE65 immunoreactivity in panel A. (D) Magnified view of CD68 immunoreactive ectopic RPE in panel B. (E-G) Human liver tissue. E RPE65 positive control. F Sham control for RPE65. G CD68 positive control, blue arrowheads indicate CD68+ Kupffer cells. (H) Sham control for CD68. d, soft drusen; BLamD, basal laminar deposit; NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner unclear layer; HFL, Henle fiber layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE, retinal pigment epithelium; BrM, Bruch's membrane; Ch, choroid; Sc, sclera.