Fig. 3

The effect of Did B treatment on F-actin and eEF1A distribution in HEp-2 cells. a-c HEp-2 cells were treated with Did B at 2.5 nM, DMSO or untreated as indicated. Fixed cells stained with Alexa Fluor 647 phalloidin or an anti-eEF1A antibody that was counter stained with a FITC conjugated secondary antibody. All images were acquired using identical settings and show a maximum intensity projection. Boxes highlight accumulation of F-actin at the cortex. Circles highlight examples of stress fibers. The arrows highlight regions of the plasma membrane with increase co-localization of eEF1A and F-actin, which is less evident in the control samples. Representative maximum intensity projection images are shown from an experiment performed three times with similar results. d The colocalization coefficient of eEF1A and actin at the cell cortex for untreated cells or those treated with DMSO or 2.5 mM Did B is shown. The cortex regions of at least 12 cells were measured. ZEN Blue Black software was used to analyze FITC (eEF1A) and phalloidin (actin) pixels using identical parameters. Colocalization analysis was performed on a pixel by pixel basis of z-stacks of images [19]. The correlation coefficient mean value and SD is shown. The **** symbols indicates a P values < 0.0001