JCS03031 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Transient overexpression of ORC1 in 293T cells does not activate the ATM/Chk2 DNA damage checkpoint pathway. (A) 293T cells transfected with FLAG-ORC1 and acute selected were analyzed as in Fig. 2B. (B) Cell cycle analysis of 293T cells transfected with FLAG-ORC1. (C) Whole cell lysates were prepared from 293T cells that had been exposed to 1.0 or 5.0 Gy γ-irradiation (IR) or treated with 2.5 mM hydroxyurea (HU), and subjected to immunoblotting. (D) The signal intensities of the bands displayed in (C) were quantified, and shown with untreated 293T cells set at 1.

• Supplemental Figure 2 -

  Fig. S2. γ-H2AX nuclear foci and NBS1 phosphorylation in 293T cells transiently transfected with Cdt1. The 293T cells were transiently transfected with T7-Cdt1 as described in Fig. 2A, and then subjected to immunostaining with anti-γ-H2AX antibody (A) and immunoblotting with anti-phospho-NBS1 (serine 343) antibody (B).

• Supplemental Figure 3 -

  Fig. S3. Synchronization and cell cycle progression of parental Rat-1 cells. Rat-1 cells were arrested in quiescence, released, harvested at the indicated time points, and analyzed by flow cytometry and immunoblotting as described in Fig. 4A and B.

• Supplemental Figure 4 -

  Fig. S4. Induction of T7-Cdt1 expression in quiescent 3Y1 cells induces chromosomal damage and ATM phosphorylation without replication. By introducing pTet-on and pTRE-Tight-T7-Cdt1 Cy (Clontech), we established a 3Y1 clone in which overexpression of Cy mutant T7-Cdt1 is inducible by doxycycline. The Tet-on T7-Cdt1 Cy 3Y1 cells were first arrested in quiescence by contact inhibition and serum starvation. Expression of T7-Cdt1 Cy was then induced by doxycycline (2μg/ml) in the quiescent cells, and simultaneously BrdU was added. After 48 hours post-induction, cells were analyzed. (A) Whole cell lysates were immunoblotted. (B) Cells were immunostained with anti-BrdU antibody. Asynchronous (AS) cells serve as a positive control for BrdU uptake. (C) Cells were double-immunostained with anti-T7 and anti-phospho-ATM antibodies.

• Supplemental Figure 5 -

  Fig. S5. Growth rates and cell cycle analysis of exponentially growing normal human fibroblasts overexpressing Cdt1. HFF2 cells were infected with the T7-Cdt1 retroviruses as in Fig. 6, and subjected to analysis after 2 weeks post-infection. (A) Growth rates. (B) Cell cycle analysis by flow cytometry.