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Fig. 3 | Cell & Bioscience

Fig. 3

From: Characterization of novel interactions with membrane NEU1 highlights new regulatory functions for the Elastin Receptor Complex in monocyte interaction with endothelial cells

Fig. 3

Binding of EDP to the ERC potentiates monocyte adhesion to endothelial cells through NEU1. (a, b) THP-1 monocytes were incubated, or not, with κE (50 µg/mL, 1 h), labelled with Calcein-AM, and allowed to adhere for 30 min at 37 °C onto a monolayer of HUVEC pre-stimulated, or not, with PMA (100 nM). Cells were then washed, fixed and visualized under a fluorescent microscope. a A representative field is shown. b The percentage of surface area covered by adherent monocytes was calculated for each condition from 10 different fields per experiment, each run in duplicate. Results were expressed as mean ± SEM of 4–8 independent experiments and normalized to the control condition (without, w/o) in presence of PMA. Statistical analysis was performed by Student’s t-test (***p < 0.001; ns: non-significant). c Similar experiments as in b except that HUVEC, instead of THP-1 cells, were incubated, or not, with κE (50 µg/mL, 1 h) prior to monocyte adhesion. Results were expressed as mean ± SEM of 3–4 independent experiments. Statistical analysis was performed by Student’s t-test (***p < 0.001; ns: non-significant). d THP-1 monocytes were incubated, or not, with DANA (400 µM), anti-CD18 blocking antibody (10 µg/mL) or isotype control (10 µg/mL) for 1 h at 37 °C before stimulation, or not, by κE (50 µg/mL), V14 + κE (molar ratio 2:1) or V14 peptide alone for 1 h at 37 °C. After adhesion (30 min, 37 °C), the percentage of surface area covered by adherent monocytes was calculated for each condition from 10 different fields per experiment, each run in duplicate. Results were expressed as mean ± SEM of 3–4 independent experiments, and normalized to the control condition (w/o). Statistical analysis was performed by Student’s t-test (**p < 0.01; ***p < 0.001; ns: non-significant). Black bars, non-stimulated cells; white bars, κE-stimulated cells. e Similar experiments as in d except that HUVEC, instead of THP-1 cells, were incubated, or not, with DANA (400 µM) followed by stimulation, or not, κE (50 µg/mL), V14 + κE (molar ratio 2:1) or V14 peptide alone for 1 h at 37 °C. Results were expressed as mean ± SEM of 3–6 independent experiments. Statistical analysis was performed by Student’s t-test (***p < 0.001; ns: non-significant)

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