HGF induces α4β1-mediated adhesion of DLBCL cells in a PI3K-dependent fashion. (A) HGF induces adhesion of DLBCL cell lines OCI-LY-1 and OCI-LY-10 to VCAM-1. Cells were stimulated with 200 ng/mL HGF or 50 ng/mL PMA followed by adhesion to VCAM-1. The OCI-LY-3 cells displayed extensive (constitutive) cell aggregation. Neither HGF nor PMA could enhance adhesion of OCI-LY-3 (three left panels). MET-negative OCI-LY-7 and OCI-LY-18 cells were used as negative controls (two right panels). The results are expressed as a percentage of maximal adhesion. The bars represent the means ± the standard deviation of a triplicate experiment representative of at least 3 independent experiments. (B) HGF-induced adhesion involves α4β1 integrin. The effect of preincubation with anti-α4β1 (HP2/1) and anti-α4β7 (Act-1) integrin antibodies on the HGF-induced binding of DLBCL cell lines OCI-LY-10 to VCAM-1 was established. Cells were preincubated for 30 minutes at 4°C in the presence or absence of anti-integrin monoclonal antibodies or isotype control antibody, as indicated. Next, adhesion to VCAM-1 in the presence of 200 ng/mL HGF was measured. Error bars represent the means ± standard deviation of a triplicate experiment representative of 2 independent experiments. (C) HGF-induced adhesion requires PI3K activity. HGF-induced adhesion of OCI-LY-10 was determined after pretreatment with the PI3K inhibitors wortmannin (WM, 100 nM) and LY294002 (LY, 20 μM), the MEK inhibitor PD98059 (PD, 50 μM), or DMSO (C) for 30 minutes at 37°C, followed by adhesion to VCAM-1 in the presence of 200 ng/mL HGF. The bars represent the means ± standard deviation of a triplicate experiment representative of 2 independent experiments.