Figure 7

Changes in morphology and relative changes in superoxide production, F/G-actin ratio, and actin recovery half-time following different experimental perturbations of the actin cytoskeleton. A) Ra2 LIMK1-WT80, Ra2 LIMK1-DN80, Ra2 cofilin-shRNA1, and Ra2 wild type cells treated for five minutes with 100 ng/ml latrunculin or 8 μM jasplakinolide were paraformaldehyde fixed and stained with Alexa 568-conjugated phalloidin, and then Z-stacks (20-40 planes, ca. 0.25-0.75 μm section width) were acquired. In the case of 8 μM jasplakinolide treatment z-stacks were generated using β-actin-YFP fluorescence as jasplakinolide inhibits phalloidin binding to F-actin. Laser intensity and gain was adjusted to show detail and therefore does not reflect the levels of polymerized actin between cell lines. The images shown represent a XY-view of the most ventral z-plane of the cell (center) while a XZ projection is shown at the top, and a ZY projection on the side. B) For each parameter (superoxide production, F/G-actin ratio, and actin recovery half-time) absolute values derived from experiments above with PMA-stimulated cells were normalized to that of Ra2 045 control cells and depicted on the Y-axis in arbitrary units.