Requirement for p21 in cell cycle arrest following nocodazole treatment. (A) Immunoblot analysis for p21 protein on extracts from wild-type and p53−/− MEFs treated with nocodazole. Unsynchronized cells were treated with nocodazole for times indicated and harvested for protein analysis. (B) FACS profiles of p21−/− MEFs untreated or treated with nocodazole for 24 h. DNA content is represented on the x axis; number of cells counted is represented on the y axis. Data shown are representative of three experiments performed on two different p21−/− clones. (C) Immunofluorescent staining of p21−/− MEFs. Cells were treated with nocodazole for 24 h, pulsed with BrdU in the presence of nocodazole for an additional 4 h, and fixed. Immunofluorescence was performed to detect BrdU incorporation (α-BrdU) and nuclear staining (DAPI). Immunofluorescence was performed on two different p21−/− clones. (D) Quantitation of number of cells in S phase in wild-type, p21−/−, and p53−/− MEFs following nocodazole treatment. Cells were treated with nocodazole, and immunofluorescence was performed as described for panel C. Data shown are the averages from three different experiments, with standard deviations as indicated. In each experiment, 100 cells of each genotype were examined and the number of BrdU-positive nuclei was counted to determine the percentage of S phase cells. Two different clones were tested for each genotype.