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. 2005 Apr;25(8):3364–3387. doi: 10.1128/MCB.25.8.3364-3387.2005

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Copyright © 2005, American Society for Microbiology

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FIG. 3. — Phosphorylation of p21 precedes G₂/M progression. (A) H460 cells were treated with nocodazole (0.8 μg/ml) for different time points (as indicated) and harvested. Samples were electrophoresed by using SDS-15% PAGE, and membranes were immunoblotted with different antibodies to detect p21, Cdc2, CDK2, cyclin B1, cyclin A, Cdc25C, PCNA, and actin (as indicated). Phosphorylation of p21 was observed after 6 h of nocodazole treatment, and this increased till 18 h, where equal intensities of hyperphosphorylated and hypophosphorylated bands were noted between 15 to 18 h. Gradually the hyperphosphorylated band intensity decreased at subsequent time points but persisted till 48 h. The Cdc2 and cyclin B1 band intensity became stronger after 12 h of nocodazole treatment and undetectable in the Western blots after 36 h of nocodazole treatment. The mitotic form of Cdc25C appeared after 9 h of nocodazole treatment and persisted till 36 h of nocodazole exposure. The level of CDK2 did not appear to change significantly with nocodazole treatment. PCNA expression also remained essentially unchanged throughout the prolonged period of nocodazole treatment. Actin was used as a loading control. Blots were probed with anti-Cdc2-Y15 antibodies in nocodazole-treated cells, and this revealed strong bands of inactive Y15-phosphorylated protein till 6 h after nocodazole exposure, and subsequently Cdc2 phosphorylated at Y15 diminished up to 36 h, with reappearance of a strong banding pattern by 48 h. Cell cycle distribution is depicted in the chart at the bottom of panel A. BrdU incorporation as an indicator of S phase, in nocodazole-treated H460 cells, showed complete loss of S phase with increase in G₂/M-phase cells after 18 h. Similar results were observed in cells treated with nocodazole alone compared to those with nocodazole- plus BrdU-treated cells. (B) Delayed S-G2 and G₂/M progression in p21^−/− cells versus p21^+/+ cells. Analysis of cell cycle profile after nocodazole treatment for HCT116 and DLD1 cells demonstrated that the change in profile from that of asynchronous cells took 3 h for p21^+/+ cells, whereas for the p21^−/− cells it took 6 h. FACS analysis also showed a higher number of cell deaths at longer time periods of nocodazole treatment for p21^−/− cells than for p21^+/+ cells. Cell cycle analysis is shown for the entire time course in the table below the histograms. AS refers to asynchronous cells. (C) Comparison of p21^+/+ and p21^−/− cells released from aphidicolin block at different time points showed appearance of p21 phosphorylation as early as 3 h and maximum phosphorylation at 6 to 8 h after release, coinciding well with an increased percentage of G₂/M cells. Subsequently, the phosphorylated p21 disappeared as the cells progressed to the subsequent cell phase.

FIG. 3. — Phosphorylation of p21 precedes G₂/M progression. (A) H460 cells were treated with nocodazole (0.8 μg/ml) for different time points (as indicated) and harvested. Samples were electrophoresed by using SDS-15% PAGE, and membranes were immunoblotted with different antibodies to detect p21, Cdc2, CDK2, cyclin B1, cyclin A, Cdc25C, PCNA, and actin (as indicated). Phosphorylation of p21 was observed after 6 h of nocodazole treatment, and this increased till 18 h, where equal intensities of hyperphosphorylated and hypophosphorylated bands were noted between 15 to 18 h. Gradually the hyperphosphorylated band intensity decreased at subsequent time points but persisted till 48 h. The Cdc2 and cyclin B1 band intensity became stronger after 12 h of nocodazole treatment and undetectable in the Western blots after 36 h of nocodazole treatment. The mitotic form of Cdc25C appeared after 9 h of nocodazole treatment and persisted till 36 h of nocodazole exposure. The level of CDK2 did not appear to change significantly with nocodazole treatment. PCNA expression also remained essentially unchanged throughout the prolonged period of nocodazole treatment. Actin was used as a loading control. Blots were probed with anti-Cdc2-Y15 antibodies in nocodazole-treated cells, and this revealed strong bands of inactive Y15-phosphorylated protein till 6 h after nocodazole exposure, and subsequently Cdc2 phosphorylated at Y15 diminished up to 36 h, with reappearance of a strong banding pattern by 48 h. Cell cycle distribution is depicted in the chart at the bottom of panel A. BrdU incorporation as an indicator of S phase, in nocodazole-treated H460 cells, showed complete loss of S phase with increase in G₂/M-phase cells after 18 h. Similar results were observed in cells treated with nocodazole alone compared to those with nocodazole- plus BrdU-treated cells. (B) Delayed S-G2 and G₂/M progression in p21^−/− cells versus p21^+/+ cells. Analysis of cell cycle profile after nocodazole treatment for HCT116 and DLD1 cells demonstrated that the change in profile from that of asynchronous cells took 3 h for p21^+/+ cells, whereas for the p21^−/− cells it took 6 h. FACS analysis also showed a higher number of cell deaths at longer time periods of nocodazole treatment for p21^−/− cells than for p21^+/+ cells. Cell cycle analysis is shown for the entire time course in the table below the histograms. AS refers to asynchronous cells. (C) Comparison of p21^+/+ and p21^−/− cells released from aphidicolin block at different time points showed appearance of p21 phosphorylation as early as 3 h and maximum phosphorylation at 6 to 8 h after release, coinciding well with an increased percentage of G₂/M cells. Subsequently, the phosphorylated p21 disappeared as the cells progressed to the subsequent cell phase.