Fig 1: Human plasma and mouse serum studies Chemokine expressions in the BOECs treated with autologous plasma. Fold changes in gene expressions of CXCL10, CXCL11, CXCL12, CCL20, and CX3CL1 in NAFLD (n = 10) BOECs were normalized to healthy (n = 12) BOECs. Results are indicated by mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001; ns—not significant (Mann–Whitney test).Plasma concentrations of CXCL10 and CXCL12 in healthy and NAFLD subjects. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers). ns—not significant (Mann–Whitney test).Serum CXCL12 concentrations of wild-type C57BL/6J mice placed on LIDPAD or control diet for 12 and 16 weeks (n = 5 mice), determined by ELISA. Data presented as box-whisker plots of median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers). ns—not significant (two-way ANOVA).
Fig 2: Characterization of cells expressing transmembrane chemokines and their receptors in proliferative vitreoretinopathy epiretinal fibrocellular membranes. Immunohistochemical stainings for CXCL16 (A), CXCR6 (B), CX3CL1 (C), and CX3CR1 (D) showing immunoreactivities in spindle-shaped myofibroblasts. Double immunohistochemistry for CD45 (brown) and CXCL16 (red) (E) or CX3CL1 (red) (F) showed co-expression in leukocytes (arrows). No counterstain to visualize the cell nuclei was applied in panels E and F (scale bar, 10 µm).
Fig 3: Human retinal microvascular endothelial cells (HRMECs) express CXCL16, CXCR6, CX3CL1, and CX3CR1. HRMECs were left untreated or treated with interleukin-1 beta (IL-1ß) (50 ng/ml) or tumor necrosis factor-alpha (TNF-a) (50 ng/ml) for 24 h. Protein expression of CXCL16 (A), CXCR6 (B), CX3CL1 (C), and CX3CR1 (D) in cell lysates was determined by Western blot analysis (representative Western blots are depicted on top of the graphs). The same loading control (ß-actin) was used for quantitation of the relative band intensity of both CXCL16 and CXCR6. Levels of CX3CL1 were quantified in the culture media by ELISA (E). The box plots (median and interquartile range) show results from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney tests were used for comparisons between three groups and two groups, respectively. *P < 0.05 compared with values obtained from untreated cells. #p < 0.05 compared with IL-1ß-treated cells.
Fig 4: Characterization of cells expressing CXCL16 and CX3CL1 in proliferative diabetic retinopathy epiretinal fibrovascular membranes. Immunohistochemical staining for CXCL16 (A) and CX3CL1 (C) showing immunoreactivity in vascular endothelial cells (arrows). Immunoreactivity for CXCL16 was also detected in stromal spindle-shaped cells (arrows) (B). Double immunohistochemistry for CD45 (brown) and CXCL16 (red) (D) or CX3CL1 (red) (E) demonstrated co-expression in stromal leukocytes (arrows). No counterstain was applied in panels (D, E) (scale bar, 10 µm).
Fig 5: Validation of chemokine hallmarks in human endothelial cells and mouse disease models Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20, and CX3CL1 in NAFLD (n = 10) BOECs, normalized to healthy (n = 12) BOECs, with 2 technical replicates per donor. Results are indicated by mean ± SD. *P < 0.05; **P < 0.01; ns—not significant (Mann–Whitney test).Left: Representative images showing nontreated and LPS + FFA-treated NAFLD and healthy BOECs, stained with Nile Red (scale bars, 100 µm). Right: Fluorescence intensity of intracellular Nile Red stain was quantified at 515/585 nm, and readings were normalized with blanks (n = 3 donors/group). Results are indicated by mean ± SD. ns—not significant (two-way ANOVA comparing NAFLD and healthy groups).Fold change in gene expressions of CXCL10, CXCL11, CXCL12, CCL20, and CX3CL1 in LPS + FFA-treated or nontreated NAFLD (n = 5) and healthy (n = 5) BOECs, normalized to nontreated healthy BOECs, with 3 technical replicates per donor. Results are indicated by mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (two-way ANOVA).Protein concentrations of chemokines in the conditioned culture media of healthy and NAFLD BOECs. *P < 0.05; ns—not significant (Mann–Whitney test). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers).Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of liver sections from chow diet-fed and HFHC diet-fed humanized mice (scale bar, 40 µm). Boxed regions were further magnified (scale bar, 30 µm). Right: Box-whisker plot of Mander’s coefficient (M2) that was a measure of the degree of CXCL12 overlap with CDH5-positive vasculatures in the livers. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers). Chow diet-fed (n = 4) and HFHC diet-fed (n = 4) humanized mice were analyzed, with 10–20 independent regions of interest per mouse were analyzed for image quantification. ****P < 0.0001 (Mann–Whitney test).Left: Representative images (Bright field; DAPI, nuclei; CDH5, endothelial cells; CXCL12) of aortic sections from chow diet-fed and LIDPAD mice (scale bar, 40 µm). Boxed regions were further magnified (scale bar, 30 µm). Arrows point at aortic endothelial cells. AW—aortic wall; L—lumen. Right: Box-whisker plot of CXCL12 fluorescent intensity in the aortic vascular endothelia. CXCL12 fluorescent intensity for each delimitated endothelial region is represented as individual data points on the plot. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers). Chow diet-fed (n = 3) and LIDPAD diet-fed (n = 3) mice were analyzed, with 10–20 delimitated endothelial regions per mouse analyzed for image quantification. ****P < 0.0001 (Mann–Whitney test).Left: Representative images showing aortic sections harvested from mice fed with either chow or LIDPAD diet. Sections were stained for leukocyte markers CD45 (red) to determine the extent of leukocyte infiltration. Dotted lines demarcate the aortic walls (AW). L—lumen. Scale bar: 20 µm. Right: Box-whisker plot of mean fluorescence intensity (MFI) of CD45 signals as a readout for the amount of infiltrated CD45+ leukocytes within the aortic walls. Box-whisker plots indicate median (middle line), 25th, 75th percentile (box), and the lowest/highest data points (whiskers). n = 3 mice per group, with 2 tissue sections analyzed per animal. **P < 0.01 (Mann–Whitney).
Supplier Page from Abcam for Human Fractalkine ELISA Kit (CX3CL1)