Fig 1: Galectin-3 retards macrophage accumulation and invasion in vitro and in vivo. Representative images of Matrigel-coated invasion Transwell inserts and quantification of in vitro macrophage invasion of (A) Lgals3-/- and Lgals3+/+ mouse monocyte-derived macrophages and (B) Lgals3-/- mouse monocyte-derived macrophages following the addition of mouse recombinant galectin-3 (5 nM); n=6/group; ***P<0.001; 2-tailed Student t test. C, Representative CD (cluster of differentiation)-68 immunohistochemical-labeled images and quantification of macrophage density in an in vivo subcutaneous sponge invasion assay performed in Lgals3-/- and Lgals3+/+ male mice; n=6/group; ***P<0.001; 2-tailed Student t test. Scale bar in A equates to 50 µm and applies to all panels in A and B, and scale bar in C equates to 100 µm. Arrows in A and B depict macrophages (CD68-positive cells).
Fig 2: RNA sequencing and gene expression analysis of galectin genes in ES cells and XEN: (a) heat map of RNA sequencing of transcripts in ES cells and XEN showing Lgals genes 1–4, 7–9, and 12 and genes involved in O-GlcNAcylation Ogt, Mgea5, Gfpt1, and Gfpt2; data are shown for 3 biological replicates denoted as -A, -B, and -C; (b) expression levels of Lgals1, Lgals2, Lgals3, Lgals4, Lgals7, Lgals8, Lgals9, and Lgals12 as detected by RT-qPCR. Bars represent mean ± SEM, N = 3.
Fig 3: Galectin-3 antagonizes generation of a proinflammatory macrophage phenotype. LGALS3 (lectin, galactoside-binding, soluble, 3) mRNA expression was silenced for 48 h in human monocyte-derived macrophages, scrambled AllStars oligonucleotides served as a control. A, Quantitative polymerase chain reaction (qPCR) for MMP (matrix metalloproteinase)-1, 2, 8, 10, 11, 12, 14, and 25 mRNA expression; n=4/group; *P<0.05; 2-tailed Student t test. B, Representative Western blot images and relative quantification of MMP12 protein levels in human monocyte-derived macrophages; n=4/group; ***P<0.05; 2-tailed Student t test. Stain-free control is shown as a loading control (LC). Real-time qPCR for TIMP (tissue inhibitor of metalloproteinase) mRNA (C), proinflammatory markers (D), and anti-inflammatory markers (E); n=4/group; *P<0.05 and **P<0.01; 2-tailed Student t test.
Fig 4: Galectin-3 regulates macrophage TGF (transforming growth factor)-ß signaling and dampens polarization of proinflammatory macrophages and their accumulation within atherosclerotic lesions. A, Real-time quantitative polymerase chain reaction (RT-qPCR) for TGFB1 mRNA expression and (B) Western blotting for active TGFß1 protein expression, in human monocyte-derived macrophages after LGALS3 gene silencing; n=4/group; *P<0.05; 2-tailed Student t test. C, Representative Western blot image and relative quantification of phospho-SMAD3 (mothers against decapentaplegic homolog 3) protein levels in human monocyte-derived macrophages after LGALS3 gene silencing; n=4/group; **P<0.01; 2-tailed Student t test. A free-stain control is shown as a loading control (LC) in both B and C. RT-qPCR for MMP (matrix metalloproteinase)-12 (D), CCL2 (chemokine [C-C motif] ligand 2; E), and PTGS2 (prostaglandin-endoperoxide synthase 2; F) in human monocyte-derived macrophages after the addition of recombinant human TGFß1 (0.2 ng/mL) for 24 h; n=4/group; *P<0.05, **P<0.01; 2-tailed Student t test. G, Quantification of in vitro macrophage invasion assay. Monocyte-derived human macrophages were allowed to invade through Matrigel-coated insert for 48 h in the presence of mouse IgG (1 µg/mL), recombinant human TGFß1 (0.2 ng/mL), or TGFß1 blocking neutralizing antibody (NAb; 1 µg/mL), following LGALS3 (lectin, galactoside-binding, soluble, 3) silencing; n=4/group; *P<0.05 vs controls, $P<0.05 vs si-Gal-3; Kruskal-Wallis nonparametric ANOVA. H–L, Representative images and relative quantification of (H) TGFß1, (I) phospho-SMAD3, (J) MMP12, (K) CCL2, and (L) PTGS2 protein expression by immunohistochemistry in Lgals3-/-:Apoe-/- and Lgals3+/+:Apoe-/- male mouse brachiocephalic atherosclerotic lesions after 8 wk of high-fat feeding; n=13/group; *P<0.05, **P<0.01; 2-tailed Student t test. Scale bar in H equates to 200 µm and applies to panels in H and J–L, and scale bar in (I) equates to 100 µm.
Fig 5: Galectin-3 deficiency in Apoe-/- mice promotes an advanced plaque phenotype. Representative images and relative quantification of (A) macrophages (CD [cluster of differentiation]-68), (B) necrotic core (hematoxylin and eosin [H&E]; area encapsulated by dotted lines represents necrotic core), (C) vascular smooth muscle cell (VSMC; aSMactin [alpha-smooth muscle actin]), and (D) collagen (picrosirius red) from Lgals3-/-:Apoe-/- and Lgals3+/+:Apoe-/- male mouse brachiocephalic atherosclerotic lesions after 8 wk of high-fat feeding; n=13/group; *P<0.05, **P<0.01; 2-tailed Student t test. E, Representative images of elastin/van Gieson and quantification of plaque vulnerability index of Lgals3-/-:Apoe-/- and Lgals3+/+:Apoe-/- mouse brachiocephalic lesions; n=13/group; ***P<0.001; 2-tailed Student t test. Scale bar in A equates to 100 µm and applies to all panels.
Supplier Page from Abcam for Mouse Galectin 3 ELISA Kit