Fig. 2.

p53 is required for p21Cip1 expression and cell cycle arrest in the absence of Ras signaling. (A) Colony formation of Rasless and K-Raslox MEFs transfected with the indicated cDNA or shRNAs and expressed as the ratio between the number of colonies observed in cells lacking Ras proteins (Rasless) and those cells expressing K-Ras (K-Raslox). The knockdown efficiency of the shRNAs is indicated on the right side of the graph. Data are represented as mean ± SD. (B) Time-lapse imaging of Rasless cells stably expressing the FUCCI cell cycle indicators mKO2-hCdt1 (red) and mAG-hGeminin (green) infected with lentiviruses expressing a control shRNA (Upper) or shp53-A (Lower). Arrows indicate two daughter cells generated from a Rasless cell that reentered the cell cycle. Time after infection (in hours) is shown in the upper right corner of each photograph. (Scale bar, 50 μm.) (C) Schematic representation of the events shown in B. (D) Western blot analysis of p53, p53 phosphorylated at serine 18 (P-p53Ser18), murine double minute 2 (Mdm2), and K-Ras expression in K-Raslox MEFs left untreated or treated with 4OHT for the indicated times. K-Raslox MEFs treated with 5 μg/mL doxorubicin (D) for 24 h were used as a positive control. GAPDH expression served as a loading control.