Figure 4

Effects of GRM1 expression on PMN adhesion and migration in vitro. (A) HMEC-1 cells were grown to confluence in 96-well black culture plates and exposed to 24 hour conditioned medium from NS and GRM1-silenced MDA-MB-231 cells or LACZ and GRM1-overexpressed MDA-MB-468 cells for 30 minutes or to TNFα (10 ng/ml) for 6 hours as a positive control. The medium was removed and BCECF-AM labeled PMNs (see Methods section) were added at 2 × 10⁵/well and allowed to attach for 30 minutes. Unattached PMNs were removed by washing in PBS and adhered PMNs detected by FITC fluorescence. (B) HMEC-1 cells were grown to confluence on BD cell culture inserts (8 μm pore size) in 24-well plates. Medium in bottom of wells were replaced with 24 hour conditioned medium as described above and labeled PMNs were added (2 × 10⁶ per insert) and allowed to migrate. After 1.5 hours, medium was removed and PMNs isolated by centrifugation and detected by measuring FITC fluorescence. For both (A,B), results are the mean ± SEM of n = 3 experiments, performed in triplicate where *is P < 0.05 compared to endothelial monolayers treated with their respective NS or LACZ conditioned medium.