Figure 5

U73343 alters MTX-induced membrane blebbing. BAECs cultured on circular glass coverslips were visualized using an inverted microscope equipped for epifluorescence as described in Materials and Methods. The cells were maintained at 35–37°C and both phase and fluorescence images were acquired at 30-second intervals. The graph in each panel shows normalized single cell fluorescence intensity as a function of time for cells pretreated with either 0.2% DMSO (Panel A; n = 38) or U73343 (Panel B; n = 52) and subsequently challenged with MTX at the times indicated. Merged phase and fluorescence images (red pseudocolor) at time 10, 25, and 40 min are shown above each graph. Examples of large membrane blebs are highlighted by the blue arrows in Panel A at time 25 min. Examples of cells with rounded morphology are indicated by the blue arrows in Panel B at time 40 min. Note the absence of large dilated blebs and lack of fluorescence in the U73343-treated cells (Panel B). Results shown are representative of 9 and 6 independent experiments for Panels A and B, respectively.