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Essential role of human CDT1 in DNA replication and chromatin licensing

DRO-Oncology, Pharmacology Department, Pharmacia Corp., Via Pasteur 10, 20014 Nerviano, Italy

View larger version (30K): [in a new window]   Fig. 1. Characterization of anti-Cdt1 antibodies. (A) 10 µg of protein extracts prepared from U2OS cells transiently transfected with either empty vector (lanes 1,3) or with plasmid expressing hCdt1 (lanes 2,4) were probed with anti-Cdt1 serum in the absence (lanes 1,2) or presence of competitor peptide (lanes 3,4). (B) 50 µg of protein extracts obtained from NHDF were blotted with anti-Cdt1 serum in the absence (lane 1) or presence (lane 2) of competitor peptide. The arrow indicates hCdt1 protein. (C) 50 µg of protein extracts from U2OS cells transfected with Cdt1-expressing plasmid (lanes 1,4), or from U20S (lanes 2,5) or HeLa (lanes 3,6) cells probed with immunopurified anti-Cdt1 antibodies in the absence (lanes 1-3) or in presence of competitor peptide (lanes 4-6).

View larger version (37K): [in a new window]   Fig. 2. Analysis of licensing factors upon re-entry into cell cycle. NHDF cells were arrested in G0 and stimulated with 10% serum to enter cell cycle. Samples were taken at the indicated times after serum stimulation. (A) DNA content was measured by FACS. (B) Total cell extracts were prepared and hCdt1, geminin, Cdc6 and cyclin A protein levels were analyzed by western blot.

View larger version (32K): [in a new window]   Fig. 3. hCdt1 function is essential for DNA replication initiation. (A) Nocodazole treated, G2/M arrested U2OS cells were microinjected with anti-Cdt1 (black bars) or anti-Cdt1 antibodies neutralized with antigen peptide (dashed bars). Microinjected cells (white arrows) also express GFP. 20 hours after nocodazole release in the presence of BrdU, cells were fixed and scored for BrdU incorporation. The graphs summarize the number of BrdU-positive cells in each sample. The number of scored cells is indicated. Original magnification x63. (B) HU-treated, S-phase arrested cells were injected as in A. Cells were released from HU into fresh medium for 20 hours. BrdU was present for the first 7 hours after HU release. Original magnification x630.

View larger version (26K): [in a new window]   Fig. 4. hCdt1 is required for the loading of MCM2 onto chromatin. (A) U2OS cells were synchronized either at G1/S border with aphidicolin (5 µg/ml) for 24 hours or at G2/M transition with nocodazole (50 ng/ml) for 16 hours as indicated. The graph summarizes the number of cells showing MCM2 nuclear staining with (black bars) or without (grey bars) Triton X-100 extraction before fixation. (B) U2OS cells were treated as in Fig. 3A. The graph summarizes the percentage of cells showing MCM2 chromatin binding in the indicated conditions. Representative fields are shown in C. Original magnification x550.

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