Fig. 4. The formation of the ruffling lamellipod in R-Ras-expressing cells depends on PLC activity. (A) Pharmacological inhibitors of intracellular Ca²⁺ and PLC inhibit R-Ras-enhanced adhesion. To assess adhesion, control and R-Ras^38V-expressing cells were loaded with 20 µg/ml calcein-AM for 20 minutes. The cells were then pretreated with the indicated pharmacological agent (DMSO, EGTA, BAPTA or the broad-specificity PLC inhibitor, U73122) for 30 minutes, and allowed to adhere onto 96-well plates coated with 20 µg/ml fibronectin for 15 and 50 minutes. The number of adherent cells was evaluated with a fluorescent plate reader, using a standard curve to determine cell number from calcein fluorescence. (B) Inhibition of phospholipase C reverts the strong peripheral actin ring induced by expression of R-Ras^38V. Control and R-Ras^38V cells were pretreated with 1 µM of the phospholipase C inhibitor, U73122, for 30 minutes followed by adhesion onto 20 µg/ml fibronectin for 30 minutes. The cells were stained with Alexa-phalloidin to visualize F-actin. (C) Inhibition of phospholipase C diminishes cell spreading. The area of cells prepared as in B was analyzed with Image J, as in Fig. 3. Treatment of both control and R-Ras38V-expressing cells with U73122 inhibits the measured area of cell spreading. Data represent measurements of >20 cells and are plotted as a box-and-whisker plots ± s.d. ^***P<0.001.