Loss of cortactin causes an ICAM-1–dependent reduction of leukocyte transmigration. (A) Human primary neutrophils were allowed to transmigrate through a TNF-activated monolayer of HUVECs on transwell filters (5-µm pore size) transfected with either control (ctrl) or cortactin-specific siRNAs. (B) Transmigration of mouse PMNs through a TNF-activated monolayer of MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice on transwell filters (3-µm pore size). The amount of transmigrated neutrophils is displayed as the percentage of total applied cells. (C) Micrographs of HUVECs transfected with control and cortactin siRNA or of MLECs from Cttn^WT/WT (WT) and Cttn^del/del (KO) mice, grown on transwell filters parallel to the experiments shown in A and B and stained for VE-cadherin. Bar, 20 µm. (A–C) Experiments were independently performed three times. (D) ICAM-1–deficient (KO) and ICAM-1–retransfected (retr.) endothelioma cells were transfected with either control or cortactin-specific siRNAs. 48 h later, cell lysates were immunoblotted for cortactin expression (top). Equal loading was controlled by blotting for tubulin (bottom). (E) Bone marrow–derived murine PMNs were allowed to transmigrate across TNF-activated ICAM-1–deficient (KO) and ICAM-1–retransfected endothelioma cell lines transfected with either control or cortactin-specific siRNA. (D and E) Experiments were performed three times. (F) Transmigration of human PMNs through HUVEC monolayers transfected with either control or cortactin-specific siRNA and additionally treated with either 100 µM 8-pCPT-2Me-cAMP (+007) or vehicle (−007). (A, B, E, and F) All data are means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.005 (n = 6 in three independent experiments; n.s., not significant).