Figure 4.

NFAT1‐induced IL8 expression promotes migration of human neutrophils. A, Primary human neutrophils were treated either with recombinant human IL8 (rhIL8; 10 ng/ml, 20 min) or with serum‐free conditioned media collected from either untreated MDA‐MB‐231 cells (231 CM) or cells in which the overexpression of NFAT1 was induced (231 NFAT1 CA CM; doxycycline 200 ng/ml, 48 h). Where indicated IL8‐neutralizing antibody was added (IL8 NAb; 50 μg/ml, 10 min). B, neutrophils were preactivated with fMLP (10 μM, 10 min), pretreated with IL8‐neutralizing antibody (NAb) or S6K1 inhibitor PF‐4708671 (5 μM), and added to transwell chambers in migration assays for 1 h towards serum‐free media (Blank), recombinant IL8 (rhIL8), or conditioned media collected from untreated MDA‐MB‐231 cells (231 CM), or cells in which the overexpression of NFAT1 had been induced (231 NFAT1 CA CM; doxycycline 200 ng/ml, 48 h). C, immunoblotting of IL8 in conditioned media (CM) collected from MDA‐MB‐231 cells transduced with NFAT1 CA with and without IL8 shRNA. D, neutrophils pre‐treated with fMLP as above were added to Transwell chambers and migrated towards recombinant human IL8 (rhIL8; 10 ng/ml), or conditioned media collected from the conditions in C. Graphs show mean numbers of neutrophils per 20× view ±SD. E, neutrophils were treated with serum‐free DMEM, rhIL8 (final concentration 1 ng/ml), or conditioned media collected after 2‐h incubation from MDA‐MB‐231 cells that were either untreated or treated with doxycycline (200 ng/ml, 48 h) to induce expression of active NFAT1 (final dilution of conditioned media 100×). Extracellular reactive oxygen species (ROS) was measured immediately afterwards and recorded over the indicated period of time and normalized to negative control. Statistical significance was determined by Student's unpaired t‐test. *p < 0.05, **p < 0.01; ***p < 0.001. All results are representative of at least 3 independent experiments.