Simultaneous inhibition of Rap1 and p38 MAPK or PI3K inhibition reversed LPS induced block of F-actin polarization and chemotaxis in moncoytes.

<p><b>A</b>) Monocytes in RPMI with 5% FBS were treated with 20 µM Rap1 inhibitor GGTI-298 (Calbiochem, EMD Biosci Corp), 10 µM 42/44ERK MAPK inhibitor PD98059 (Cell signaling), 10 µM p38 MAPK inhibitor SB203580 (Tocris Corp), 20 µM GGTI-298+10 µM PD98059 and 20 µM GGTI-298+10 µM SB203580, or DMSO control for 30 min at 37°C. Cells were plated on cover slips and treated with or without LPS (2 ng/ml) at 37°C for 15 min, followed by stimulation with 20 nM CCL2 for 2 min. Cells were fixed and stained with phalloidin-488, and images were collected with Leica TCS SP5. <b>B</b>) 10⁷ monocytes were treated with inhibitors as described above for 30 min followed by a 15 min incubation with or without (control) LPS (2 ng/ml). ∼0.5×10⁶ LPS treated or untreated cells in 100 µl of RPMI with 1% FBS were loaded in the upper Transwell chambers (Nunc 5.0 µm) challenged with or without 20 nM CCL2 in the bottom chambers, and incubated at 37°C CO₂ incubator for 2 h. Chemotaxis was measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030404#s4" target="_blank">Methods</a>. Migrated cells for control and LPS treatments after respective inhibitors are plotted pair wise in the histograms (with error bars). n = 3, ^***p<0.01,^**p<0.04, ^*p<0.05. <b>C</b>) LPS induced Rap1 activation was inhibited in monocytes pretreated with GGTI-298 alone or with GGTI-298 and p38 MAPK inhibitor, SB203580, but not with SB203580 alone. Activated (GTP+) Rap1 was extracted from cytoplasmic extracts using a commercial pull-down kit followed by SDS/PAGE and imunoblotting with anti-Rap1 mAb. Numbers represent relative fraction (%) of Rapi that was activated (n = 3). <b>D</b>) PI3K inhibitors reversed LPS induced block of F-actin polarization in monocytes stimulated with CCL2. Monocytes in 5%FBS/RPMI were treated with 50 µM LY2940002, 1 µM wortmannin, or DMSO control for 30 min 37°C. Cells were plated on cover slips and treated with or without LPS (2 ng/ml) at 37°C for 20 min, followed by 20 nM CCL2 for 2 min. Cells were fixed and stained with phalloidin-488, and images were collected with Leica TCS SP5.