Figure 2

AmpA influences the level of polymerized F actin. A) Wild type,amp A knockout (KO) and overexpresser (OE) cells werestained with Alexa 488-phalloidin to detect F-actin (green) and TRITClabeled DNAse I to detect G-actin (red). Imaging conditions wereoptimized for levels of phalloidin staining (see Methods). The fields ofcells from which the representative individual cells were excised are inAdditional file 5. The images are 3Dreconstructions from a confocal z series. Scale bar is 20 um. Thefluorescent F-actin images were all equally enhanced to more clearly seethe F-actin in the ampA knockout. B) Fields of Wt, KOand OE cells stained with Alexa 488 phalloidin shown in Additional file5 were quantified (see Methods). Theaverage pixel intensity per cell, cell surface area and pixelintensity/um² are shown. Significance was determined by atwo-tailed paired Student’s t-test with a p value < 0.05indicated as significantly different from Wild type. 15 to 20 cells perstrain were quantified per experiment. The staining experiment wasrepeated 6 different times with cells grown on different days. C)The amount of Alexa 488-phalloidin bound to cells was determined (seeMethods). Results are averages of 3 different experiments with datapoints in triplicate. Error bars are standard error of the mean.