Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Depletion of Geminin by siRNA induces rereplication. Western blot (A) and phase-contrast microscopic images (B) of HCT116 cells treated with Geminin or control (GL2) siRNA for 48 h. Western blots were probed with antibodies specific for Geminin and Vinculin (loading control). (C) Flow cytometric profiles of HCT116 treated with Geminin siRNA for 48 h. Before harvesting, the cells were pulsed with BrdU for 2 h and processed for combined propidium iodide staining together with cyclin B1– and BrdU-specific antibodies. The black bars in the phase-contrast pictures correspond to 10 μm. (D) Summary of the protocol used in Geminin siRNA–treated U2OS cells to measure DNA rereplication within a single cell cycle. (E) Geminin expression levels in U2OS cells released from nocodazole block and treated with GL2 (control) or Geminin siRNA. (F) Distribution of [ 3 H]thymidine-labeled DNA in the CsCl gradient fractions. U2OS cells were processed according to the protocol described in A. DNA was prepared and subjected to CsCl density gradient centrifugation as described in Materials and methods. Y axes: [ 3 H]thymidine radioactivity in cpm; x axes: number of gradient fraction collected from the bottom. The fractions containing LL (light-light), HL (heavy-light), and HH (heavy-heavy) DNA were determined by a series of control experiments (not depicted).

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Western Blot, Control, Staining, Expressing, Blocking Assay, Labeling, Gradient Centrifugation, Radioactivity

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rescue of Geminin siRNA phenotype in cells stably expressing HA-tagged RNAi mutants of Geminin. U2OS cell lines expressing two different mutant HA-tagged versions of Geminin were generated (Geminin-Δi179 and Geminin-Δi179-2). In the cDNA for Geminin, two residues were changed in the siRNA target sequence in a way that the nucleotide sequence, but not the amino acid sequence, was altered (C). The cell lines were treated with Geminin siRNA for 48 h and analyzed by Western blot with antibodies specific for the indicated proteins (A) and by FACS ® analysis for DNA content (B). Note the decrease in cells with DNA content greater than and equal to 4N.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Stable Transfection, Expressing, Mutagenesis, Generated, Sequencing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Geminin depletion activates the checkpoint response during S phase. U2OS cells were transfected with siRNA to Geminin or control and synchronized in mitosis by nocodazole treatment for 16 h. The cells were subsequently released into the next cell cycle and retransfected with Geminin or control siRNA. The cells were harvested at the indicated time points and analyzed by Western blot using the indicated antibodies (A), and for DNA content by FACS ® analysis (B). (C) Cells grown on the coverslips were labeled with BrdU for 10 min at each time point, pre-extracted with 0.5% Triton X-100 in cytoskeleton buffer, fixed with PFA, and immunostained for BrdU and chromatin-bound MCM2. (D) Kinetics of DNA damage checkpoint induction and progression through mitosis of Geminin- or control siRNA–treated cells. The cells were plated on poly- d -lysine–coated coverslips, fixed, and stained with phosphohistone H3 and γH2AX antibodies. 70 cells were counted for each time point.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Transfection, Control, Western Blot, Labeling, Staining

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rereplication induced by Geminin depletion is dependent on CDT1 and CDC6. HCT116 cells were treated with control (GL2), Geminin, CDT1, and CDC6 siRNA at the indicated combinations for 48 h. Cells were harvested and analyzed for protein expression (A), DNA content (B), and cell proliferation (D). (C) Phase-contrast microscopic images taken of cells treated for 48 h with the siRNA for the indicated transcripts. Note the rescue of giant nuclei formation formed in Geminin siRNA–treated cells when cotransfected with CDC6 or CDT1 siRNAs. (E) Model for the role of Geminin in controlling origin firing in S phase. Rereplication results in the activation of an ATR-CHK1–dependent checkpoint that results in block of entry into mitosis. Even though rereplication leads to DNA strand breaks (e.g., phosphorylation of H2AX and formation of RAD51 nuclear foci) and subsequent p53 activation, functional p53 does not appear to be involved in either regulation of rereplication or the DNA damage checkpoint response.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Control, Expressing, Activation Assay, Blocking Assay, Phospho-proteomics, Functional Assay

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rereplication induced by Geminin depletion activates CHK1 and a DNA damage response. (A) Western blot analysis of cellular extracts prepared at the indicated time points from HCT116 cells treated with Geminin or control (GL2) siRNAs. S317CHK1, Y15CDC2, and S15p53 indicate the use of antibodies specifically recognizing the phosphorylated amino acid of the proteins. (B) Rereplicated cells contain H2AX and Rad51 nuclear foci. U2OS cells treated with control (GL2) or Geminin siRNA for 48 h were immunostained with rabbit polyclonal H2AX and RAD51 antibodies. (C) Rereplicated cells contain ssDNA coated by RPA70. HCT116 cells were prelabeled with 10 μM BrdU for 24 h, incubated with Geminin or control (GL2) siRNA for 48 h, fixed with methanol, and immunostained with a BrdU-specific antibody without denaturation of DNA. BrdU foci (green) correspond to the sites of ssDNA breaks. RPA immunostaining is shown in red. (D) Formation of giant nuclei and CHK1 activation in TIG3 human diploid fibroblasts treated with Geminin siRNA. TIG3 cells were transfected twice with GL2 (control) or Geminin siRNA, fixed, and stained 96 h after the first transfection.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Western Blot, Control, Incubation, Immunostaining, Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Loss of p53 is not required for rereplication or DNA damage response. (A) U2OS cells were infected with virus generated by pRetroSuper (pRS) p53 producing short hairpin RNA against p53 or with empty virus. Cells were subsequently treated with Geminin-specific siRNA for 48 h and analyzed by Western blotting and FACS ® analysis. Western blots were probed with antibodies specific for the indicated proteins. Actinomycin D treatment (1 nM) was used as a positive control for p53 activation. (B) HCT116 cells infected with empty virus or E6-expressing virus were treated and analyzed as in A.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Infection, Virus, Generated, shRNA, Western Blot, Positive Control, Activation Assay, Expressing