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

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Spontaneous colony formation in AMPKα1-KO MEFs. Wild type (WT), AMPKα1-KO, and AMPKα2-KO MEFs (1 × 10 5 cells/mL) were seeded and cultured on 6-well plates. Culture medium was changed every 2 days for 3 weeks. (Upper) Representative images showing colony formation of MEFs. (Bottom) Quantification of colony formation. n =5, * P <0.001 versus WT. B. Anchorage-independent cell growth assay (soft agar assay) of MEFs. (Upper) Representative images for colony formation. Scale bar =500 μm. (Bottom) Quantification of colony formation. n =10, * P <0.001 versus WT. C. Phosphorylated CDK2 at The-160 (pCDK2-T160) and CDK2 are upregulated in AMPKα1-KO MEFs. pCDK2-T160 and CDK2 protein in WT, AMPKα1-KO, and AMPKα2-KO MEFs were analyzed by Western blot (top). Quantification of pCDK2 and CDK2 data (bottom). n =4, * P <0.01 versus WT; † P <0.05 versus WT. D. Diminished anchorage-independent growth of AMPKα1-KO MEFs following CDK2 knockdown by shRNA. Representative images are shown. Scale bar =500 μm. E. Representative Western blot data indicate CDK2 knockdown by shRNA (top). Quantification of anchorage-independent MEF growth (bottom). n =4, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Culture, Growth Assay, Soft Agar Assay, Western Blot, Knockdown, shRNA, Control

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Upregulation of CDK2 transcription in AMPKα1-KO MEFs. CDK2 mRNA levels were measured by quantitative reverse transcription polymerase chain reaction in WT and AMPKα1-KO MEFs. n =5, * P <0.01 versus WT. B. p52 is responsible for CDK2 elevation in AMPKα1-KO MEFs. MEFs were transfected with either control siRNA or p52 siRNA (100 nmol/L) for 72 hours. Representative blot from three independent experiments (top). Quantification of Western blot data (bottom). n =3, * P <0.01 versus WT/control siRNA; † P <0.05 versus α1-KO/control siRNA. C. Chromatin immunoprecipitation (ChIP) analysis of the CDK2 gene. MEF chromatin from WT and AMPKα1-KO mice was immunoprecipitated with anti-p52 or rabbit IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for mouse CDK2 promoter. This result is representative of four independent experiments. n =4, * P <0.05 versus WT. D. p52 knockdown by shRNA diminishes anchorage-independent growth of AMPKα1-KO MEFs. Representative images are shown. Scale bar =500 μm. E. Quantification of anchorage-independent MEF growth. Data are mean ± SD, n =5, * P <0.01 versus WT/control shRNA; † P <0.01 versus α1-KO/control shRNA.

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Transfection, Control, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Amplification, Knockdown, shRNA

Journal: Oncotarget

Article Title: AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

doi: 10.18632/oncotarget.10687

Figure Lengend Snippet: A. Plasma concentrations of Epo in nude mice inoculated with WT or AMPKα1-KO MEFs. n =10-13 per group; * P <0.01 versus WT. B. Epo upregulation in inoculated AMPKα1-KO MEFs in nude mice. Implanted MEF tissues were collected from nude mice euthanized by carbon dioxide inhalation at 6 weeks after MEFs implantation. Representative images show Epo staining (top). Quantification of anti-Epo staining (bottom). n =5 per group; * P <0.01 versus WT. C. Tumor growth rate of AMPKα1-KO MEFs in nude mice injected intraperitoneally (IP) with an antibody specific to Epo or IgG. n =8–10 per group, † P <0.01 versus WT/IgG; * P <0.01 versus α1-KO/IgG. D. Representative images showing staining with antibodies specific to CD-31 or smooth muscle (SM)-α-actin (left). Scale bar =50 μm. Quantification of anti-CD31or SM-α-actin staining (right). Implanted AMPKα1-KO MEFs were collected from nude mice euthanized by carbon dioxide inhalation at the end of experiment (8 weeks after MEFs implantation). n =5–6 per group; * P <0.01 versus IgG. E. Mechanisms of AMPKα1deletion-stimulated anchorage-independent growth, neovascularization, and consequent tumorigenesis. AMPKα1 deletion in fibroblast activates NIK, which phosphorylates and activates IκKα, then enhance p100 phosphorylation recruiting E3 ubiquitin ligase β-TrCP, which facilitates p100 processing to p52. Upregulated p52 controls CDK2 and Epo expression. Epo-mediated neovascularization cooperating with CDK2-mediated anchorage-independent growth contributes to tumorigenesis in vivo .

Article Snippet: The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): rabbit anti-CDK2 (2546), rabbit anti-pCDK2-T160 (2561), rabbit anti-pp100-S866/870 (4810), rabbit anti-pIĸKα/β (2078), rabbit anti-IĸKα (2682), rabbit anti-NF-ĸB2 p100/p52 (4882), rabbit anti-NIK (4994), rabbit anti-β-TrCP (D13F10) (4394), and rabbit anti-PCNA (13110). β-TrCP siRNA (sc-37179), p52 siRNA (m) (sc-36043), CDK2 shRNA (m) lentiviral particles (sc-29260-V), NFκB p52 shRNA (m) lentiviral particles (sc-36043-V), goat anti-pNIK-T559 (sc-12957), rabbit anti-Epo (sc-7956), mouse anti-GAPDH (sc-32233), mouse anti-β-actin (sc-47778), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Clinical Proteomics, Staining, Injection, Phospho-proteomics, Ubiquitin Proteomics, Expressing, In Vivo

Journal: Journal of Biological Chemistry

Article Title: C/EBPα Is Required for Proteolytic Cleavage of Cyclin A by Calpain 3 in Myeloid Precursor Cells

doi: 10.1074/jbc.m204096200

Figure Lengend Snippet: FIG. 6. Truncated cyclin A is capable of forming active com- plexes with cdk2. A, Western blot of cyclin A protein in nuclear extracts of C/EBP wild type (/), heterozygous (), and null (/) newborn liver after incubation in vitro. The positions of full-length and 38-kDa cleaved cyclin A (A38) isoforms are shown to the right. The blot was reprobed with -actin as a control. B, kinase assay for cyclin A-associated cdk2 activity using HH1 as a substrate. Cyclin A was immunoprecipitated (cyc A IP) from incubated newborn liver extracts of C/EBP/, , and / mice (shown in A). Mock immunoprecipita- tions (using protein A-agarose beads with no antibodies; ag. con) were used as a control for specificity. After SDS-PAGE, proteins were trans- ferred to a nitrocellulose membrane and subjected to autoradiography and phosphorimaging, and then the membrane was stained with Coo- massie Blue to verify equal loading of the HH1 substrate (lower panel). Phosphorylated HH1 (*HH1) is shown in the upper panel. C, diagram of the human cyclin A protein. The N-terminal destruction box (D-box) is shown as well as the cyclin box involved in cdk binding and activation. The site of C/EBP-dependent calpain 3 cleavage is shown (amino acids 120–130).

Article Snippet: Polyclonal antibodies specific for the C terminus of cyclin A (C-19), C/EBP (14-AA), cdk2 (M-2) or the histidine (His) epitope (G-18) are all from Santa Cruz Biotechnology.

Techniques: Western Blot, Incubation, In Vitro, Control, Kinase Assay, Activity Assay, Immunoprecipitation, SDS Page, Membrane, Autoradiography, Staining, Binding Assay, Activation Assay