Fig. 3. Endogenous R-Ras contributes to cell spreading. MCF10A cells were transfected with siRNA oligonucleotides of a random sequence (control) or targeted against R-Ras. (A) siRNAs directed against R-Ras are effective. Half of the transfected cells were lysed, and equal cell numbers for control and R-Ras siRNA separated by SDS-PAGE. Transfers were immunoblotted with antibodies against R-Ras to demonstrate a significant knockdown with the anti-R-Ras siRNA. (B) Cell spreading is diminished by R-Ras knockdown. The other half of the cells transfected were detached and allowed to spread on 20 µg/ml fibronectin for 45 minutes, fixed, and cell area was analyzed as described in the Materials and Methods using Image J software. Results for 20 cells are graphed in a box-and-whisker plot such that the horizontal line represents the mean, the box represents the 25% and 75% confidence intervals, and the error bars represent the s.d. ^***P<0.001 compared with cell area of control cells using a two-tailed unpaired t-test.