Fig. 5

eCIRP induces neutrophils to acquire JAM-C and VE-cadherin from endothelial cells and become pro-inflammatory. A The neutrophils trans-migrated through the endothelial cell layer on the transwell membrane (TBMN) and freshly isolated neutrophils (BMN) were further analyzed by immunostaining with antibodies to anti-JAM-C (red color), anti-VE-cadherin (magenta color), and anti-CD11b (yellow color). The neutrophils stained with antibodies were mounted on the glass slide and images were acquired with Zeiss LSM900 Airyscan 2 mode. The neutrophils were pre-stained with Celltrace Violet dye and shown in blue color in the pictures and endothelial cells were stained with PE (membrite) shown in green color prior to the assay. The arrow in the merged panel indicates co-localization of the signals. Scale bar, 2 µm. B The trans-migrated neutrophils (TBMN) stained as in the A were observed individually under the confocal microscope. JAM-C-positive TBMN treated with eCIRP were compared to the PBS control in the TEM assay. C The ICAM-1 expression level was observed by confocal microscopy. The trans-migrated neutrophils were collected and stained with anti-JAM-C antibody and anti-ICAM-1 antibody and the cells were individually imaged. The expression level of ICAM-1 in JAM-C positive neutrophils were compared to the level of ICAM-1 in JAM-C negative neutrophils. Data represented as mean ± SEM. *p < 0.05, unpaired, two-tailed student’s t-test