Fig 1: NGF-eGFP Reporter Activity following Calvarial Bone InjuryNGF-eGFP reporter animals were subjected to a circular, full-thickness frontal bone defect (1.8 mm in diameter).(A–E) µCT reconstructions of the defect site in a top-down view (left) and coronal cross-sectional images (right) among (A) uninjured frontal bones and (B–E) d3–d28 post-injury. Margins of original defect are indicated by dashed black or red lines. Black scale bar, 500 µm; white scale bar, 200 µm.(F–J) Representative tile scans (left) and high-magnification images (right) of NGF-eGFP reporter activity in the calvarial defect site at serial time points, including (F) uninjured frontal bones and (G–J) d3–d28 post-injury. Reporter activity appears green, while nuclear counterstain appears blue. Uninjured control is shown for comparison. White arrowheads indicate margins of defect site. Dashed white lines indicate healing bone edge. White scale bar, 50 µm.(K) Quantification of relative NGF-eGFP reporter activity from d3–d28 post-injury.(L–N) Immunohistochemical staining (appearing red) within the defect area of NGF-eGFP reporter animals, including (L) PDGFRa (platelet-derived growth factor receptor a), d3 post-injury; (M) F4/80, d3 post-injury; and (N) osteocalcin (OCN), d14 after injury. In graphs, each dot represents a single animal; n = 3 animals per time point. White scale bar, 50 µm. Data are represented as mean ± SD.
Fig 2: Effects of the dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin on oxidized low-density lipoprotein accumulation and its related gene expression levels in macrophages extracted from diabetic db/db mice and db/db misty mice at 13 weeks old. Four days after an intraperitoneal injection of thioglycolate, the exudate peritoneal cells were isolated from diabetic db/db (C57BLKS/J Iar -+Leprdb/+Leprdb) mice and db/db misty (C57BLKS/J Iar -m+/m+) mice at 13 weeks of age. For cholesterol accumulation, adherent macrophages were incubated at 37°C in 5% CO2 for 18 hours with RPMI 1640 medium containing 5% fetal bovine serum (FBS) and 10 µg/mL oxidized low-density lipoprotein (ox-LDL) in the presence of 0.1 mmol/L [3H] oleate or 10 µg/mL Dil-ox-LDL, which were added with/without 10 nmol/L teneligliptin (Te) ex vivo (a-k). For gene expression levels, adherent macrophages were incubated at 37°C in 5% CO2 for 18 hours with RPMI 1640 medium with 5% FBS in the presence/absence 10 nmol/L teneligliptin ex vivo without the addition of ox-LDL (l-n). (a-i) Representative immunofluorescent staining images of the peritoneal macrophages isolated from diabetic db/db mice or db/db misty mice. F4/80-expressing cells were in green, Dil-ox-LDL-stained cells were in red, and DAPI-stained cells were in blue. Scale bars represent 50 µm. (j) Relative fluorescence ratio per area of Dil-ox-LDL in peritoneal macrophages isolated from mice was imaged and analyzed with a BZ-X710 microscope/software. (k) Foam cell formation in exudate peritoneal macrophages isolated from diabetic db/db mice and db/db misty mice. The cellular lipids were extracted, and the radioactivity of the cholesterol [3H] oleate was determined by thin-layer chromatography. (l-n) Gene expression levels related to foam cell formation in peritoneal macrophages isolated from diabetic db/db mice and db/db misty mice. Gene expression levels of CD36 (l), acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) (m), and interleukin-6 (IL-6) (n) and the association with GAPDH were analyzed by real-time RT-PCR without the addition of ox-LDL. Data information: n = 7db/db misty mice, n = 7db/db mice, and n = 7db/db mice with ex vivo teneligliptin treatment. Results are presented as mean values ± SEM and analyzed with one-way ANOVA: ??? p < 0.005, ?? p < 0.01 vs. db/db misty mice. ??? p < 0.005, ?? p < 0.01, ? p < 0.05 vs. db/db mice without teneligliptin.
Fig 3: Immune landscape of the prostate includes a prostate-specific macrophage subset enriched in metallothionein transcripts(A) UMAP of 793 cells in myeloid compartment after integration of myeloid/MNP cells from n = 10 patients with Henry et al. myeloid/MNP cells.(B) Mean expression dot plot of top five significant marker genes for each myeloid cluster. Marker genes were identified using Wilcoxon rank sum test and p adj < 0.05 was considered statistically significant. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value.(C) UMAP plot of predicted MNP clusters in prostate cancer single cell data from (Karthaus et al., 2020, Chen et al., 2021, Crowley et al., 2020).(D) (top) Representative RNAscope images of probes targeting MT1 family genes (magenta) and CD68 (yellow). ‘L’ indicates lumen. Arrows point to single cells that are marked by both probes in sub-panels i and ii. Scale bar, 20 µm. (bottom) Representative immunofluorescence microscopy images of a human prostate section labeled for metallothionein (a-MT)/isotype control (yellow), HLA-DR (cyan), CD206 (purple) and DAPI (blue). White arrows point to structure displaying colocalization of a-MT with HLA-DR and/or CD206 labeling. Scale bars, 50 µm.(E) Mean expression dot plot of Zinc transporter genes for each myeloid cluster. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value.(F) Heatmap of mean AUCell enrichment of F4/80hi/lo gene sets, corresponding to yolk sac (YS) versus hematopoetic stem-cell (HSC) lineage. Row enrichment value is scaled from 0 to 1 and presented as an increasing gradient from black, gray, yellow to orange which corresponds to increasing enrichment score.(G) Representative immunofluorescence microscopy images of cross sections of mouse prostate labeled for F4/80 (green), MHCII (red), CD11b (blue), CD31 (yellow) and phalloidin. Scale bars, 120 µm.(H) Cell counts per gram of prostate for rat IgG2a isotype or anti-Csf1r antibody (Ab) treated male mice. N = 5 per group. ****p < 0.0001; n.s denotes not significant (p > 0.05) (Two-way ANOVA with Tukey’s multiple correction).(I) Zinc concentration of anterior prostate lobe, liver lobe, and kidney from male mice treated with either rat IgG2a isotype control or anti-Csf1r Ab. N = 6 per group. (shown is representative quantification from one of two independent experiments). *p < 0.05; n.s. not significant (Mann-Whitney test). See also Figures S4–S5.
Fig 4: Lymphoid single-cell landscape of normal prostate and prostate cancer(A) UMAP of 1694 lymphoid cells from n = 7 patients. Expression of marker genes for NK cells (FCGR3A, GNLY), CD8 T cells (CD8B), tissue residency and activation (CD69) and cytolytic molecule (GZMA) are shown as a heatmap where gray indicates no expression and increasing expression is colored from purple, orange to yellow.(B) Dot plot of top five significant marker genes for each lymphoid clusters. Marker genes were identified using Wilcoxon rank sum test and p adj < 0.05 was considered statistically significant. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to blue corresponds to increasing expression value.(C) Pie chart showing proportion of cells expressing markers for (left) memory (CD27+IGHD-), naive (IGHD+CD27-), non-naive (remainder) and (right) heavy gene constant gene expression.(D) Confocal imaging of CD19, IgG and CD31 in normal murine prostate section. Scale bars, 50 µm.(E) (Top) Volcano plot showing top 15 significant DEGs between NK CD16pos and NK CD16neg (normal only). (Bottom) Violin plots of gene set testing (AUCell) for NK cell gene sets (KEGG and GO) and lymphocyte tissue residency gene sets from (Mackay et al., 2016). Significance is denoted by **p < 0.01; ***p < 0.001 (Mann-Whitney test). Position of asterisks indicate the group with higher expression.(F) Confocal imaging of NKp46, MHCII, F4/80 and CD31 in normal murine prostate section. Scale bars, 35 µm. See also Figure S6.
Fig 5: Effects of SMTP-44D on Dil-ox-LDL uptake into, and RAGE, Cdk5, and CD36 gene expression in, Apoe-/- mice. Peritoneal macrophages were extracted from Apoe-/- mice injected with SMTP-44D at 30 mg/kg/day or saline every other day for 4 weeks. (A–F) Representative immunofluorescent staining images in peritoneal macrophages. Dil-ox-LDL positive cells were stained in red (A,D), while F4/80 were in purple (B,E). (C,F) Merge images. Scale bars, 50 µm. Quantification of fluorescence intensity in red. Dil-ox-LDL uptake was shown as a relative value compared to control mice (G). Gene expression levels of RAGE (H), Cdk5 (I), and CD36 (J) derived from Apoe-/- mice, and their correlation (K,L). Total RNAs were reverse-transcribed, and the resulting cDNAs were amplified by real-time PCR. Data were normalized by the intensity of GAPDH mRNA-derived signals and expressed as a relative to the control values. Number = 6 for each group. Error bars are standard deviation. ? p < 0.05 and ?? p < 0.01 vs. control.
Supplier Page from Abcam for Anti-F4/80 antibody [F4/80] (Alexa Fluor® 647)