Fig 2: PAK inhibition leads to an accumulation of active RhoA and phosphorylated myosin light chain.(A) Human neutrophils adherent on fibronectin surfaces were pretreated with vehicle (0.1% DMSO) or PF3758309 (PF; PAK inhibitor, 10 µM), and stimulated with fMLP (10 nM) for 3 min and stained for phospho-PAK1/2 Thr423/402 (green) and F-actin (red). (B) The mean percentage of cells displaying phospho-PAK1/2 immunofluorescence at basal level (white bars) or after stimulation (black bars) was quantified from at least 50 cells per treatment. (C) Cell circularity was analyzed using MATLAB and presented as aspect ratio of individual neutrophils at basal level (white bars) or after fMLP stimulation (black bars). (D) Neutrophils were pretreated as indicated in (A) and stimulated with fMLP (10 nM). Cells were stained for active RhoA-GTP (green) and F-actin (red). (E) The mean relative intensity of RhoA-GTP immunofluorescence or (F) the mean percentage of RhoA-GTP/actin colocalization of individual cell area at basal level (white bars) or after fMLP stimulation (black bars). (G) Neutrophils were pretreated with the inhibitors indicated in (A), or with Y27632 (ROCK inhibitor, 10 µM), or a combination of 10 µM PF3758309 and 10 µM Y27632 (PF+Y27632) and stained for phospho-myosin light chain (p-MLC; green) and F-actin (red). (H) Relative p-MLC fluorescence intensity or (I) the distance between DIC cell centroid and p-MLC fluorescence centroid at basal level (white bars) or after fMLP stimulation (black bars). Representative images obtained from at least 3 independent experiments are shown. * P<0.05 compared to the basal level; # P<0.05, compared to DMSO-treated cells; ** P<0.05, compared to cells treated with Y27632 alone. Scale ba = (A) and (G) 5 µm; (D) 10 µm.