Attenuated cell spreading in Rras^−/− DCs. (A) Day 6 BM-DCs from Rras^+/+ (n = 1) or Rras^−/− (n = 1) mice were plated on fibronectin (FN; 10 μg/mL) or BSA-coated plates and allowed to attach for 20 minutes. Images were taken from 5 random fields, and the percentage of spread cells (red arrows) was scored. (B) Results are from a single experiment using increased concentrations (▴) of BM-DCs and have been repeated once with similar results. Bars represent SD. (C) Immunofluorescence analysis of day 9 Rras^+/+ (n = 1) or Rras^−/− (n = 1) BM-DCs using Texas Red-conjugated phalloidin (TR-Ph; ii-iii,viii-ix, red) to stain F-actin filaments. Cell nuclei were visualized by 4,6-diamidino-2-phenylindole (iv-vi,x-xii, blue). Arrowhead indicates spread cells. (D) The percentages of spread cells were quantified as described in “Cell spreading assay.” ns indicates not significant. (E) The maximal cell length was determined by ImageJ software, with each data point representing the mean cell length for a single field of approximately 62 cells. (C-E) Results were from a single experiment, and 9 fields were selected from triplicate wells with an average of 550 cells being analyzed per group. Bars represent SE. ***P < .0005. **P < .005. *P < .05.