Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 1. Genetic ablation of p53, Cdk2, or Cdk4 leads to absence of the respective protein expression. (A) PCR-based genotyping. Results of PCR analysis of genomic liver DNA from E.13.5 embryos generated by crossing p53/ Cdk2/ or p53/ Cdk4/ mice are shown. These gels included five double mutants (p53/ Cdk2/ [left panel] or p53/ Cdk4/ [right panel]), one Cdk2/ mutant, one Cdk4/ mutant, wild-type (Wt) embryos, and a control lacking DNA (H2O). M, molecular size marker; KO, knockout. (B) To confirm the genotyping data generated in panel A, Western blotting was performed using antibodies specific to p53, Cdk2, and Cdk4. -Actin was used as a loading control (bottom). (C) Western blotting was conducted to determine the expression levels of Cdk4 in Cdk2/ MEFs and of Cdk2 in Cdk4/ MEFs. To ensure that equal amounts of proteins were loaded, -actin was used to probe the same membrane.

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques: Expressing, Generated, Mutagenesis, Control, Marker, Knock-Out, Western Blot, Membrane

Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 2. Ablation of Cdk2 or Cdk4 does not significantly alter the cell cycle. (A and B) Cells of the indicated genotypes were arrested in G0 and subsequently stimulated by addition of serum. Cells were pulse-labeled with BrdU 30 min prior to harvest and were harvested at the indicated time points after serum stimulation. Cells were stained with anti-BrdU antibodies and the appropriate secondary antibodies and were visualized using confocal microscopy and a 63 objective. Nuclei were counterstained with DAPI. This experiment was repeated twice; results of a representative experiment are presented. Frequencies represent BrdU-positive cells in a population of at least 200 cells per group. Wt, wild type. (C) Whole-cell extracts were prepared from MEFs collected at the indicated time points following serum addition and were analyzed by Western blotting using antibodies against cyclin A, cyclin D1, cyclin E, and -actin as a control. Cyc, cyclin. (D) Western blotting was performed with MEFs cultured in DMEM containing 0.2% FBS for 48 h. The numbers 1 and 2 above the lanes represent the loading of the protein lysates of two independent MEFs of the indicated genotypes. Western blots were probed with antibodies against p21Waf1, p27Kip1, p57Kip2, and p16INK4A. -Actin served as a loading control. (E) Western blots of proteins extracted from controls (wild-type MEFs) or of wild-type MEFs transfected with siRNAs specific to p53 were probed with p53, p57, p16, and -actin (control).

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques: Labeling, Staining, Confocal Microscopy, Western Blot, Control, Cell Culture, Transfection

Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 3. Ablation of Cdk2 and Cdk4 or siRNA-mediated silencing of Cdk2 and Cdk4 leads to distinct centrosome cycle defects. (A) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, processed, and coimmunostained with anti-pericentrin, anti--tubulin, and the appropriate secondary antibodies. Averages standard deviations of percentages of cells with one, two, and three centrosomes are shown. Exactly 8 wild-type (Wt), 3 Cdk2/, 4 Cdk4/, and 3 Cdk2/ Cdk4/ embryos were analyzed. The statistical significance of the averages (P 0.05) was established by an unequal-variance t test. t test values for the percentages of cells in each population containing one centrosome relative to that for the wild type were 0.159885 for Cdk2/ MEFs, 0.000518 for Cdk4/ MEFs, and 0.000544 for Cdk2/ Cdk4/ MEFs. The P values for the percentages of cells in each population containing two centrosomes relative to that of wild-type MEFs were 0.122182 for Cdk2/ MEFs, 0.000172 for Cdk4/ MEFs, and 0.000528 for Cdk2/ Cdk4/ MEFs. The P values for the percentages of cells with 3 centrosomes relative to that of wild-type MEFs were 0.091487 for Cdk2/ MEFs, 0.06122 for Cdk4/ MEFs, and 0.000808 for Cdk2/ Cdk4/ MEFs. (B) MEFs of the indicated genotypes were grown in duplicate to confluence, followed by serum starvation for 60 h. Quiescent cells were stimulated with serum, and every 4 h for a period of 16 h, the numbers of cells with one and two centrosomes were scored. This experiment was repeated twice; results of a representative experiment are presented. The BrdU data are the same as those presented in Fig. 2A and B; they are shown here for purposes of clarity. (C) Western blots of proteins extracted from nontransfected wild-type cells, or from cells transfected with siRNAs against Cdk2 or Cdk4, and probed with antibodies against Cdk2 or Cdk4. The same membrane was probed with -actin as a control. (D) Wild-type MEFs

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques: Western Blot, Transfection, Membrane, Control

Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 4. Ablation or siRNA-mediated silencing of Cdk2 and Cdk4 prevents centriole reduplication and centrosome amplification in p53/

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 5. Ablation of Cdk2 and Cdk4 inhibits chromosome instability in cells lacking p53. (A and B) The frequencies of -H2AX foci (arrows) in cells with the indicated genotypes were calculated. Bars, 10 m. The graph (B) shows the average percentage of -H2AX foci in each population of at least 200 cells. Error bars, standard deviations. Each group included 4 different MEFs. P values (relative to the wild-type control) were 0.015218 for p53/ MEFs, 0.126173 for p53/ Cdk2/ MEFs, and 0.346771 for p53/ Cdk4/ MEFs. (C and D) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, and nuclei were visualized with DAPI. Frequencies of micronucleus (insets and arrows) formation were calculated for at least 500 cells of each of the indicated genotypes. Each group included 4 different MEFs. P values (relative to the wild-type control) were 0.016122 for p53/ MEFs, 0.137054 for p53/ Cdk2/ MEFs, and 0.370282 for p53/ Cdk4/ MEFs.

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques: Control

Journal: Molecular and Cellular Biology

Article Title: Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical Mediators of Centrosome Amplification in p53-Null Cells

doi: 10.1128/mcb.00253-09

Figure Lengend Snippet: FIG. 8. Model explaining how the ablation of Cdks prevents cen- trosome amplification. (A) Ablation of p53 results in undetectable levels of p21Waf1, leading to hyperactive Cdk2 and Cdk4. Hyperactive Cdks cross talk to the centrosome in two modes. First, hyperactive Cdks hyperphosphorylate Rb in the nucleus, leading to uncontrolled E2F-dependent transcription of molecules that influence various steps in the centrosome duplication cycle: those involved in centriole split- ting, as well as centriole duplication kinases (CtDKs). Second, hyper- active Cdks constitutively phosphorylate NPMT199, resulting in exces- sive licensing of centrosome duplication. Uncontrolled expression of CtDKs and the inability of NPM to suppress normal centrosome du- plication lead to faster centrosome duplication cycles within a single cell cycle, resulting in the formation of multiple centrosomes. (B) When Cdk2 or Cdk4 is deleted from p53/ MEFs, Rb is under- phosphorylated, and the E2F-dependent transcription of CtDKs is restored. In addition, underphosphorylated NPM restores normal cen- trosome licensing and prevents excessive centriole duplication. This restricts the centrosome duplication cycle to one per cell cycle, thus resulting in normal centrosome numbers.

Article Snippet: Cdk2 and Cdk4 small interfering RNAs (siRNAs) (catalogue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotechnology, Inc. Three independent MEFs of each genotype were grown in a six-well plate until they became 60 to 80% confluent.

Techniques: Expressing