JCS041889 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effects of changing the Cdt1 expression levels on re-replication induction in three human cell lines. HFF2/T, 293T and HeLa cells were infected with retroviruses expressing wild type (WT), T29A or Cy+D1m T7-Cdt1 or control retroviruses at different MOI (MOI=0.1, 1 and 10 for 293T and HFF2/T and MOI=0.1 and 1 for HeLa), and selected with hygromycin B. At 4 days after infection, cells were analyzed. (A) DNA content was analyzed by flow cytometry and the percentages of re-replicated cells (DNA content higher than 4N) are shown. (B) Whole cell lysates were immunoblotted with anti-Cdt1 and anti-actin antibodies. The signal intensities of the Cdt1 proteins were quantified, normalized to the signals of actin, and shown with the value of endogenous Cdt1 in HFF2/T cells set at 1.

• Supplemental Figure S2 -

  Fig. S2. Silencing of ATR and/or ATM by retroviral expression of the shRNAs does not enhance Cdt1-induced re-replication in HFF2/T cells. Cells were co-infected with the three different recombinant retroviruses as indicated, or their control empty vectors (MOI=10 for Cdt1, and MOI=1 for shRNAs). Cells were then co-selected with hygromycin B and puromycin. At 4 days after infection, cells were collected and analyzed. (A) DNA content was analyzed by flow cytometry. (B) Whole cell lysates were immunoblotted with the indicated antibodies. The membranes were also subjected to CBB staining to show equal loading.

• Supplemental Figure S3 -

  Fig. S3. Simultaneous deregulation of Cdt1+CDC6 or Cdt1+ORC1 induces re-replication and triple overexpression synergistically induces strongest re-replication in 293T cells. Cells were transfected and analyzed 48 hours after transfection as for Fig. 2. (A) DNA contents were analyzed by flow cytometry and the results shown in Fig. 2A as dot plots of FL2-A and FL2-W are demonstrated here as histograms of FL2-A. (B) The cell number of each transfectant was counted and is shown with the value for the control transfectant set at 100. The means and SDs from two independent experiments are given. 3

• Supplemental Figure S4 -

  Fig. S4. The CDC6 AAA mutant deficient in Cdk phosphorylation induces stronger re-replication than the wild type when coexpressed with Cdt1 in 293T cells. Cells were transfected and analyzed 48 hours after transfection as in Fig. 3. DNA contents were analyzed by flow cytometry and the results shown in Fig. 3A as dot plots are demonstrated here as histograms of FL2-A.

• Supplemental Figure S5 -

  Fig. S5. Simultaneous deregulation of Cdt1+CDC6 or Cdt1+ORC1 induces re-replication and triple overexpression synergistically induces strongest re-replication in normal human HFF2/T fibroblasts. Cells were co-infected with recombinant retroviruses and analyzed at 4 days after infection as in Fig. 4. DNA contents were analyzed by flow cytometry and the results shown in Fig. 4A as dot plots are demonstrated here as histograms of FL2-A.