DOI-induced rolling of AML14.3D10 cells involves activation of ROCK, MAPK PI3K, PKC and calmodulin.

<p>(<b>A</b>) Phosphorylation of p-44/42 MAPK in cells exposed to DOI after pre-treatment with PD98059 (10 µM, MAPK inhibitor) or DMSO (vehicle for inhibitor) for 5 min. Cell lysates were analyzed by Western blot analysis. (<b>B</b>) DOI-induced rolling of AML14.3D10 cells on rh VCAM-1-coated cover-slips under conditions of flow <i>in vitro</i> after treatment with MDL-100907 (5-HT2A antagonist), Y27632 (ROCK inhibitor), PD98059, LY294002 (PI3K inhibitor) (left panel) or PT (G_αi inhibitor), BIM(I) (PKC inhibitor), TFP (calmodulin inhibitor) (right panel). Cells treated with DMSO alone served as the control. All inhibitors were used at a concentration of 10 µM, except PT which was used at 100 ng/ml, for 20 min and then exposed to DOI for 5 min prior to infusion into the flow chamber. (<b>C</b>) Total F-actin measured by flow cytometry after FITC-phalloidin staining of AML14.3D10 cells treated with MDL-100907, Y27632, PD98059, LY294002 or vehicle for 20 min and then exposed to DOI. Results are expressed as percent change in mean fluorescence intensity (MFI) relative to vehicle-treated cells. (<b>D</b>) FITC-phalloidin staining and confocal microscopy of AML14.3D10 cells adhered to rh VCAM-1-coated cover-slips in the presence of MDL-100907, Y27632, PD98059, LY294002 or vehicle and then exposed to DOI (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054840#pone-0054840-g001" target="_blank">Figure 1</a>). Magnification ×600. Combined data (Mean ± SEM) of four independent experiments in B and three independent experiments in C is shown. *p<0.03 for comparison of cells exposed to DOI versus vehicle. **p<0.01 and ^#p<0.05 for comparison of cells exposed to DOI in the presence of inhibitor versus cells exposed to DOI in the presence of vehicle. Data shown in D is representative of two independent experiments.