Figure 3.

p14ARF depletion affects cell morphology. (a) Contrast images of HeLa cells transiently transfected with the indicated stealth siRNAs for 48 h, detached by trypsinization and re-plated at a density of 1 × 10⁵/ml in six-well plates. Images were collected and analyzed by phase-contrast microscope 5 and 24 h after plating with a 20 × objective (scale bar, 30 μm). (b) To quantify the percentage of each phenotype, for each transfection point, we counted adherent and round cells in five different fields and pooled data from three to five experiments. Spreading efficiency was measured by assaying the number of adherent cells relative on total cell number (graph). Cumulative data are expressed as a mean value±s.e.m. of three independent experiments. Number of cells analyzed for each experiment: siSCR (200), siARF (200), siLUC (200). Asterisks (*) indicate statistically significant differences by unpaired two-tailed t-test between siARF and both siLUC and siSCR: *P<0.0002, whereas NS indicates non statistical significant values. (c) Confocal images of HeLa cells treated with control or specific ARF siRNA for 48 h were left to adhere and doubly stained with fluorescein isothiocyanate (FITC)-conjugated phalloidin and DAPI (scale bar, 10 μm).