Fig 1: Inflammation-targeting ability and cell-specific uptake of eNABs. (A) The expression of adhesion molecules increased in neutrophils and Neu-ABs after LPS pretreatment. (B) The retainment of adhesion molecules in eNABs inherited from Neu-ABs. (C) Representative fluorescence images showed eNABs adhered to the inflamed endothelium in vitro. Scale bars, 50 μm. (D) Quantitative analysis of the fluorescence intensity of P-selectin/ICAM-1 and MSNs/eNABs in each group (n = 3). (E) Representative images of eNABs (red) engulfed by macrophages (white)/cardiomyocytes (green) in a coculture pattern. The white arrows indicate eNABs engulfed by macrophages, and the right panel shows the high magnification of the representative images. Scale bar, 50 μm. (F) Intracellular uptake of eNABs by macrophages. Scale bar, 10 μm. (G) Cell-specific uptake of eNABs by macrophages. Representative fluorescence images of macrophages after different treatments. Scale bar, 20 μm. Data are presented as means ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) for D (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig 2: Rg3 repressed atherosclerosis and adhesion molecules in intima via regulating PPARγ/FAK signaling pathway in ApoE−/− mice. (A, B) The expression of p-FAK and PPARγ in aorta by Western blotting. (C) Representative images of ICAM-1 and VCAM-1 immunohistochemical staining of aortic root cross sections. Values are expressed as the mean ± s.d. from three independent experiments. Magnification, ×400; scale bar = 100 μm. *P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. model group; ##P < 0.01 vs. model group.
Fig 3: RvD1 inhibits CRD-induced XO expression and its activity, PP2A inactivation, NFκB activation and ICAM1 and VCAM1 expression. A, C & E. Eight weeks old C57BL/6 mice were fed with CRD in combination with and without RvD1 (10 μg/kg body weight, every 3 days by IP) or left on CD for 12 weeks, aortas were isolated and either tissue extracts were prepared and assayed for H2O2 production (A) or XO and PP2A activities (C & E). B, D, F & G. An equal amount of protein from aortic tissue extracts prepared as in panel A were analyzed by Western blotting for XO, pPP2A-C(α/β), pIκBα, pIKKα/β and pNFκB, ICAM1 and VCAM1 levels and the blots were normalized for α-tubulin or their total levels. The bar graphs represent quantitative analysis of three experiments. The values are expressed as Mean ± SD. *p < 0.05 vs CD; **p < 0.05 vs CRD.
Fig 4: Effects of E2 on the levels of adherence and angiogenic factors. (A) Representative western blot images demonstrating the expression of CD49d, ICAM1 and LFA-1 in the four groups. (B) Relative protein expression levels of CD49d, ICAM1 and LFA-1. (C) Representative western blot images demonstrating the expression of MMP2, MMP9 and VCAM1 in the placenta in the four groups. (D) Relative protein expression of MMP2, MMP9 and VCAM1. n=12 rats/group. *P<0.05, **P<0.01 and ***P<0.001 vs. control; ##P<0.01 and ###P<0.001 vs. L-NAME and L-NAME + NS. E2, estradiol; ICAM1, intercellular adhesion molecule-1; LFA-1, leukocyte function-associated antigen; MMP, matrix metallopeptidase; VCAM1, vascular cell adhesion molecule-1; L-NAME, N (omega)-nitro-L-arginine methyl ester; NS, normal saline.
Fig 5: Rg3 ameliorated HUVECs function and down-regulated expression of adhesive molecules stimulated with ox-LDL. (A) The structure of 20(R)-Rg3, molecular formula: C42H72O13, molecular weight: 785.025 g/mol (U.S. national library of medicine). (B) MTT assay of different Rg3 concentration on cell viability. (C) After treatment with Rg3, cultured HUVECs had a scratch placed in the middle of the six-well plates as a basal control (0 h); then, ox-LDL (200 µg/ml) was added for 24 h. Observed under a microscope and photographed at 0, 24 h in same position. % scratch closure = (the area at 0 h - the area at 24 h)/the area at 0 h. (D) Representative images of labeled THP-1 cells adherent to HUVECs. 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM)-labeled THP-1 showed green fluorescence. The statistical number of THP-1 cells adherent to HUVECs. (E–G) The expression of ICAM-1, VCAM-1, p-FAK, and PPAR? were determined by western blotting, the phosphorylation of FAK is expressed as the ratio of phosphorylated-FAK to total FAK, GAPDH was used as a loading control. Values are expressed as the mean ± s.d. from three independent experiments; magnification, ×100; scale bar = 400 µm. *P < 0.01 vs. control group; **P < 0.01 vs. control group; #P < 0.05 vs. ox-LDL group; ##P < 0.01 vs. ox-LDL group.
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