Fig. 9. Inhibition of Rho and ROCK activities abrogate ephrin-A5-mediated cell rounding and membrane blebbing. EphA3 293T cells (A-D) were treated with 10 µM C3 transferase exoenzyme overnight (C,D) and control cells left untreated (A,B). Parallel cultures of stimulated (+) or nonstimulated (-) cells were fixed in 4% PFA and stained with rhodamin-phalloidin. (E) Treatment with C3 transferease reduces RhoA available for ribosylation. Lysates fromEphA3 293T cells with (+) or without (-) overnight C3 transferase treatment were used for ADP ribosylation assays in the presence of radioactive ³²P-NAD. RhoA-associated radioactivity was quantified after SDS-PAGE and autoradiography (top) by densitometry. Mean and s.d. from two independent experiments are shown (bottom). (F) Cell blebbing in EphA3-293T cells, left untreated or after pretreatment with 10 µM ROCK inhibitor Y-27632 or 10 µM PI3 kinase inhibitor LY 294002 (2 hours), was monitored by time-lapse microscopy. As control, blebbing in parental nontransfected 293T cells is shown. Representative fields containing approximately 100 cells were monitored for 10 minutes before and 50 minutes after stimulation. Total and blebbing cells were counted by an observer blinded to experimental conditions. Values represent mean percentages and s.d. (three independent experiments) of cells that commenced blebbing following addition of ephrin-A5. Supplemental videos of time courses illustrating responses of EphA3-393T cells without or with Y-27632 treatment are available at: http://www.ludwig.edu.au/addinfo/Figure9F.mpg and at: http://www.ludwig.edu.au/addinfo/Figure9Finhib.mpg, respectively.

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