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Figure 7


Fig. 7. PLC{epsilon} mediates R-Ras signaling to membrane protrusions. MCF10A cells stably expressing either a control vector or R-Ras38V were transfected with control siRNA, or 50 nM of pooled siRNA directed against PLC{epsilon}. (A) Cells were harvested 72 hours later, normalized by cell number and evaluated for PLC{epsilon} knockdown by immunoblotting with anti-PLC{epsilon}. (B) PLC{epsilon} siRNA diminishes the measured area of spreading. Although the effect is not statistically significant, it does extend the mean and range of cell areas measured downward as shown by the box-and-whisker plots. (C) MCF10A cells treated with siRNA against PLC{epsilon} were allowed to adhere onto 20 µg/ml fibronectin for 45 minutes, fixed, and then stained with Alexa-phalloidin to evaluate F-actin. Arrows indicate four representative phenotypes (phenotypes A-D) observed and quantified in D. Two different fields are shown for PLC{epsilon} siRNA to show the range of phenotypes observed. Phenotypes A, B and C are similar to those quantified in Fig. 2. Note the appearance of a new phenotype, D, characterized by multiple membrane protrusions. (D) Quantification of the phenotypes labeled in B for control (pZIP) or R-Ras38V-expressing cells.





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