Loss of CD63 results in a failure of P-selectin dependent rolling and adhesion of THP-1 cells on HUVECs. HUVECs were treated with 2 rounds of 2 different siRNA oligonucleotides directed against CD63 or control siRNA. HUVECs treated with IL-4 or culture media alone were transferred to μ-slides 24 hours before flow experiment. Slides were placed under flow at 0.7 dyne/cm2, and incubated with sheep polyclonal anti-human P-selectin (25 ug/mL) or media alone followed by perfusion for 5 minutes to ensure the monolayer was intact. HUVECs were treated with PMA (100 ng/mL) or perfusion media alone for 5 minutes under flow. THP-1 cells (106/mL) were then added to the perfusion media with or without PMA and antibody and allowed to flow across the monolayer for 5 minutes. The entire movie was recorded for each condition. (A) Quantification of THP-1 adhesive interactions on HUVECs showing a significant loss of interactions in CD63 depleted cells. (B) Quantification of THP-1 firm adhesion to HUVEC monolayers. The number of THP-1 cells firmly adhered to the monolayer after the 5 minutes of perfusion was recorded for at least 5 fields of view for each condition. (C) Quantification of THP-1 firm adhesion to HUVEC monolayers treated with IL-4. ***P < .001, **P < .01 by 2-way ANOVA comparing between PMA stimulated control-siRNA treated cells and CD63 siRNA-treated cells and 1-way ANOVA comparing between control-siRNA groups with Bonferroni posttests (n = 3). Error bars represent SE of the mean.
