Journal: Virology

Article Title: Characterization of murine gammaherpesvirus 68 v-cyclin interactions with cellular cdks.

doi: 10.1016/j.virol.2005.07.014

Figure Lengend Snippet: Fig. 1. gHV68 v-cyclin binds to cdk2, but not cdk4 or cdk6. (A) Cos-1 cells were transfected with pCMV-TAG2B vector control or FLAG epitope-tagged cyclins E, D or v-cyclin and pCMV-cdk2 (left panel), pCMV-cdk6 (middle panel), or pCMV-cdk4 (right panel). In the analyses of cyclin association with cdk2 and cdk4, a cyclin D1 expression vector was used, while for the analysis of association with cdk6, a cyclin D3 expression vector was used (see Materials and methods). (B) Cos-1 cells were transfected with pCMV-TAG2B vector control or FLAG epitope-tagged v-cyclin and pCMV-cdk2 (left panel), pCMV-cdk6 (middle panel), or pCMV-cdk4 (right panel). Cells were harvested 48 h post-transfection, lysates prepared, and immunoprecipitations carried out with agarose- conjugated anti-FLAG antibodies (Sigma). Immunoprecipitates were subjected to 15% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated anti-cdk antibody. Membranes were stripped and reprobed with anti-FLAG antibody to verify FLAG-v-cyclin immunoprecipitation (panel B only). WCL, whole cell lysates (5% of that used for immunoprecipitation).

Article Snippet: Antibodies used in this study were: rabbit polyclonal anti-cdc2 (C-19, Santa Cruz Biotechnology), goat polyclonal anti-cdk2 (M2, Santa Cruz Biotechnology), goat polyclonal anti-cdk4 (C-22, Santa Cruz Biotechnology), goat polyclonal anti-cdk6 (C-21, Santa Cruz Biotechnology), rabbit polyclonal anti-pRb (C15, Santa Cruz Biotechnology), mouse monoclonal antip27Kip1 (F-8, Santa Cruz Biotechnology), rabbit polyclonal anti-cyclin D3 (H292, Santa Cruz Biotechnology), rabbit polyclonal anti-cyclin A (C-19, Santa Cruz Biotechnology), mouse monoclonal anti-FLAG-HRP (M2, Sigma), mouse monoclonal anti-h-actin-FITC conjugate (Sigma), and rabbit anti-gHV68 v-cyclin antisera (van Dyk et al., 1999).

Techniques: Transfection, Plasmid Preparation, Control, FLAG-tag, Expressing, SDS Page, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 5. Expression of cell cycle regulators in NIH 3T3 fibro- blasts following H2O2 treatment. A, cell lysates were prepared from NIH 3T3 fibroblasts at the times indicated following treatment with 250 M H2O2. The expression of cyclin D1, D3, E, A, and B, CDK2, CDK4, CDC2, p21Cip1, p27Kip1, p53 were analyzed by Western blotting. Total RNA was isolated in parallel, and the expression of p21Cip1 and actin mRNA was determined by Northern blotting. B, a wild type p21Cip1 promoter-luciferase construct was transfected into cycling NIH 3T3 cells. The transfected cells were treated with 250 M H2O2, col- lected 4 h later, and lysed for luciferase assay. The luciferase activity was normalized with -galactosidase activity specified by co-transfected pJ4-gal plasmid and expressed as the mean S.D. of at least three independent experiments each with duplicated transfection. C, lysates were prepared from p53/ MEFs at the times indicated following treatment with 250 M H2O2, and the expression of cyclin D1 and D3, p21Cip1, and p27Kip1 was analyzed by Western blotting.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Isolation, Northern Blot, Luciferase, Construct, Transfection, Activity Assay, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 6. Analysis of cyclin-dependent kinase complexes and their association with CKIs in NIH 3T3 fibroblasts following H2O2 treatment. A, cell extracts prepared from untreated NIH 3T3 and NIH 3T3 cells 4 h after 250 M H2O2 treatment were immunopre- cipitated (IP) with antibodies against CDK2, CDC2, CDK4, cyclin E, cyclin A, and cyclin B1. The precipitated complexes were examined for kinase activity using a pRB fragment or Histone H1 as substrates. B, cell extracts from untreated and H2O2-treated NIH 3T3 cells, as de- scribed above, were immunodepleted of p21Cip1 or p27Kip1 by incubation with excess amounts of mouse monoclonal p21Cip1- or p27Kip1-specific antibodies coupled to protein G-Sepharose. Supernatant immunode- pleted of p21Cip1 and p27Kip1 were Western blotted for p21Cip1, p27Kip1, cyclin A, cyclin E, CDK2, cyclin B, CDC2, cyclin D1, CDK4, and CDK6 expression. As controls, a mock immunodepletion was performed with- out antibodies.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Incubation, Western Blot, Expressing, Immunodepletion

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 9. Expression of cyclins D1, E, and K after IPTG induction in the NIH 3T3 cell lines. NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either untreated or treated with 3 mM IPTG for 24 h. Lysates derived from these IPTG-induced cells were Western blotted for ectopic cyclin D1, cyclin E, and K cyclin expression. The blots were also probed for tubulin expression, which was used as loading control. The inducible cyclins D1, E, and K were detected with specific antibodies against the FLAG tag, human cyclin E, and hemagglutinin, respectively.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Derivative Assay, Western Blot, Control, FLAG-tag

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 10. Analysis of the ability of cy- clins D1, E, and K to overcome the cell cycle arrest induced by H2O2 treatment. NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either left un- treated () or treated () with 3 mM IPTG for 24 h to induce cyclin expression. These IPTG-pretreated cells as well as the un- treated cells were then treated with ei- ther 0, 250, or 500 M H2O2, prior to col- lection for cell cycle analysis 4 h afterward. BrdUrd was added to the cells for the final 30 min of the experiment. The harvested cells were fixed and stained with propidium iodide and FITC- conjugated anti-BrdUrd antibodies (lower panels) prior to FACS analysis to meas- ure the DNA contents and to determine the cells in DNA synthesis, respectively. The percentages of cells in each cell cycle phase (G1, S, and G2/M) determined by DNA contents and stained positive for BrdUrd are indicated.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Cell Cycle Assay, Staining, DNA Synthesis

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 5. Expression of cell cycle regulators in NIH 3T3 fibro- blasts following H2O2 treatment. A, cell lysates were prepared from NIH 3T3 fibroblasts at the times indicated following treatment with 250 M H2O2. The expression of cyclin D1, D3, E, A, and B, CDK2, CDK4, CDC2, p21Cip1, p27Kip1, p53 were analyzed by Western blotting. Total RNA was isolated in parallel, and the expression of p21Cip1 and actin mRNA was determined by Northern blotting. B, a wild type p21Cip1 promoter-luciferase construct was transfected into cycling NIH 3T3 cells. The transfected cells were treated with 250 M H2O2, col- lected 4 h later, and lysed for luciferase assay. The luciferase activity was normalized with -galactosidase activity specified by co-transfected pJ4-gal plasmid and expressed as the mean S.D. of at least three independent experiments each with duplicated transfection. C, lysates were prepared from p53/ MEFs at the times indicated following treatment with 250 M H2O2, and the expression of cyclin D1 and D3, p21Cip1, and p27Kip1 was analyzed by Western blotting.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Isolation, Northern Blot, Luciferase, Construct, Transfection, Activity Assay, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 6. Analysis of cyclin-dependent kinase complexes and their association with CKIs in NIH 3T3 fibroblasts following H2O2 treatment. A, cell extracts prepared from untreated NIH 3T3 and NIH 3T3 cells 4 h after 250 M H2O2 treatment were immunopre- cipitated (IP) with antibodies against CDK2, CDC2, CDK4, cyclin E, cyclin A, and cyclin B1. The precipitated complexes were examined for kinase activity using a pRB fragment or Histone H1 as substrates. B, cell extracts from untreated and H2O2-treated NIH 3T3 cells, as de- scribed above, were immunodepleted of p21Cip1 or p27Kip1 by incubation with excess amounts of mouse monoclonal p21Cip1- or p27Kip1-specific antibodies coupled to protein G-Sepharose. Supernatant immunode- pleted of p21Cip1 and p27Kip1 were Western blotted for p21Cip1, p27Kip1, cyclin A, cyclin E, CDK2, cyclin B, CDC2, cyclin D1, CDK4, and CDK6 expression. As controls, a mock immunodepletion was performed with- out antibodies.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Incubation, Western Blot, Expressing, Immunodepletion

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 9. Expression of cyclins D1, E, and K after IPTG induction in the NIH 3T3 cell lines. NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either untreated or treated with 3 mM IPTG for 24 h. Lysates derived from these IPTG-induced cells were Western blotted for ectopic cyclin D1, cyclin E, and K cyclin expression. The blots were also probed for tubulin expression, which was used as loading control. The inducible cyclins D1, E, and K were detected with specific antibodies against the FLAG tag, human cyclin E, and hemagglutinin, respectively.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Derivative Assay, Western Blot, Control, FLAG-tag

Journal: Journal of Biological Chemistry

Article Title: H2O2 Induces a Transient Multi-phase Cell Cycle Arrest in Mouse Fibroblasts through Modulating Cyclin D and p21Cip1 Expression

doi: 10.1074/jbc.m111123200

Figure Lengend Snippet: FIG. 10. Analysis of the ability of cy- clins D1, E, and K to overcome the cell cycle arrest induced by H2O2 treatment. NIH 3T3-D1, NIH 3T3-E, or NIH 3T3-K cell lines were either left un- treated () or treated () with 3 mM IPTG for 24 h to induce cyclin expression. These IPTG-pretreated cells as well as the un- treated cells were then treated with ei- ther 0, 250, or 500 M H2O2, prior to col- lection for cell cycle analysis 4 h afterward. BrdUrd was added to the cells for the final 30 min of the experiment. The harvested cells were fixed and stained with propidium iodide and FITC- conjugated anti-BrdUrd antibodies (lower panels) prior to FACS analysis to meas- ure the DNA contents and to determine the cells in DNA synthesis, respectively. The percentages of cells in each cell cycle phase (G1, S, and G2/M) determined by DNA contents and stained positive for BrdUrd are indicated.

Article Snippet: The antibodies against p21Cip1 (M-19), p27Kip1 (C-19), CDK4 (C-22), CDK6 (C-21), CDK2 (M-2), CDC2 (C-19), cyclin D1 (HD11), cyclin D1–3 (H-295), cyclin A (C-19), cyclin E (M-20), and cyclin B1 (M-433) were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Cell Cycle Assay, Staining, DNA Synthesis

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: Specific inhibition of CDK7 by 1-NMPP1 prevents DNA synthesis (A), pRb phosphorylation (B), CDK4/6 pRb-kinase activity (C) and CDK4 activating phosphorylation (D). HCT116 WT or K7AS (K7as/as) cells were stimulated (+) or not stimulated (−) with FBS for the indicated times in the absence (−) or presence (+) of 1-NMPP1. (A) DNA synthesis was evaluated from duplicate dishes by counting the percentage of cells having incorporated BrdU during the last 30 min of stimulation. (B) Western blotting analysis with the indicated antibodies from whole-cell lysates. (C) Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), anti-cyclin D3 (D3) or anti-p21 (p21) antibodies and were assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (C). (D) The same immunoprecipitates were separated by 2D gel electrophoresis followed by immunodetection using antibodies directed against CDK4 or T172-phosphorylated CDK4. Arrows, T172-phosphorylated form of CDK4.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Inhibition, DNA Synthesis, Phospho-proteomics, Activity Assay, Western Blot, Immunoprecipitation, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: Effect of acute inhibition of CDK7 by 1-NMPP1 on CDK4 activity (A) and phosphorylation (B). (A,B) HCT116 K7AS cells were stimulated with FBS for 5 h and 1-NMPP1 was then added for 1 or 3 h. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (A), or separated by 2D gel electrophoresis followed by CDK4 immunodetection (B). In (B), T160 phosphorylation of CDK2 immunodetected from whole cell extracts is shown for comparison.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Immunoprecipitation, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Comparison

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for the indicated times in the absence (−) or presence (+) of 1-NMPP1. Cell lysates were immunoprecipitated (IP) with anti-p21, anti-cyclin D1 (D1) or anti-cyclin D3 (D3) antibodies and separated by 2D gel electrophoresis followed by p21 immunodetection. (B) CHO cells were transfected with a p21-expressing plasmid alone (left) or with plasmids encoding cyclin D3, CDK4-HA and p21 (right). Cell lysates were immunoprecipitated (IP) with anti-p21 (p21) or anti-cyclin D3 (D3) antibodies and incubated with the indicated recombinant kinases and ATP, before separation by 2D gel electrophoresis and p21 immunodetection. Arrows indicate the main phosphorylated forms of p21 as identified in . (C) Schematic representation and comparison of p21 patterns from HCT116 K7AS cells that were restimulated for 3 and 20 h ( in vivo ) and from cyclin D3-CDK4-p21 complexes produced in CHO cells and phosphorylated by recombinant CDK2-cyclin E and CDK2-cyclin A ( in vitro ). The observed p21 forms are labeled according to their number of phosphorylations and the identified phosphorylated residues (e.g., 2P 98,130 means p21 phosphorylated at both S98 and S130; X, Y, unidentified phosphorylated sites).

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Transfection, Expressing, Plasmid Preparation, Incubation, Recombinant, Comparison, In Vivo, Produced, In Vitro, Labeling

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) p21 stabilizes cyclin D3-CDK4 complexes and inhibits their activity depending on its expression level. CHO cells were transfected with plasmids encoding cyclin D3 and CDK4-HA with or without p21 at high (p21) or low (p21 1/10) expression level. Cell lysates (WCE) were immunoprecipitated (IP) with anti-cyclin D3 (D3) or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies. (B) p21 expressed alone is essentially unphosphorylated. CHO cells were transfected with a plasmid encoding p21 at high (p21) or low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-p21 antibody and separated by 2D gel electrophoresis followed by p21 immunodetection. (C) Co-expression of cyclin D3-CDK4 induces the phosphorylation of p21, largely independently of CDK4 activity, whereas stronger expression of p21 inhibits phosphorylations of both p21 and CDK4. CHO cells were transfected with plasmids encoding cyclin D3 and CDK4-HA or its inactive mutants (T172A, K35R) with or without p21 at high (p21) or low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 antibody and separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection. (D) Identification of p21 phosphorylation sites. CHO cells were transfected with plasmids encoding cyclin D3, CDK4-HA and wild type (WT) or mutated (S130A, S98A) p21 at low (p21 1/10) expression level. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 antibody and separated by 2D gel electrophoresis followed by p21 immunodetection. Black arrows, T172-phosphorylated CDK4. Colored arrows indicate phosphorylated forms of p21.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Activity Assay, Expressing, Transfection, Immunoprecipitation, SDS Page, Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Phospho-proteomics

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A,B) Stably infected HCT116 K7AS cells for Tet-On inducible 3×HA-p21 wt or S130A mutant were treated with doxycycline (1 µg/ml) for 16 h prior cell restimulation with fetal bovine serum (FBS) for 16 h in the continuous presence of doxycycline. (A) Immunodetection of 3×HA-p21 and endogenous p21 from whole cell extracts (WCE) using a p21 antibody. (B) Cell lysates were immunoprecipitated (IP) with anti-HA antibody and separated by 2D gel electrophoresis followed by simultaneous immunodetection of ectopic 3×HA-p21 and 3×HA-p21-bound endogenous CDK4 using a mixture of anti-CDK4 and p21 antibodies. Black arrows, T172-phosphorylated CDK4. Colored arrows indicate phosphorylated forms of p21.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Stable Transfection, Infection, Mutagenesis, Immunodetection, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for the indicated times in the absence (−) or presence (+) of roscovitine (rosco) (A,B) or CR8 (C). (A) Western blotting analysis with the indicated antibodies from whole-cell lysates. (B,C) Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1), anti-cyclin D3 (D3) or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunodetection of CDK4 and p21. Black arrows, T172-phosphorylated CDK4; green arrows, S130-phosphorylated p21.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Western Blot, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A,B) HCT116 K7AS cells were stimulated (+) or not stimulated (−) with fetal bovine serum (FBS) for 5 h in the absence (−) or presence (+) of the CDK4/6 inhibitor PD0332991. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1) or anti-p21 antibodies, assayed for their pRb-kinase activity, separated by SDS-PAGE and immunoblotted with the indicated antibodies (A), or separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection (B). (C) CHO cells were transfected with plasmids encoding cyclin D3, CDK4-HA and p21 at low (p21 1/10) expression level (see ), in the absence (DMSO as vehicle) or presence of CDK inhibitors: roscovitine (rosco), PD0332991 (1 µM) or their combination to inhibit both CDK2 and CDK4. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 (D3) antibody and separated by 2D gel electrophoresis followed by CDK4 and p21 immunodetection. Black arrows, T172-phosphorylated CDK4; colored arrows, phosphorylated forms of p21.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Immunoprecipitation, Activity Assay, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Immunodetection, Transfection, Expressing

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: (A) HCT116 K7AS cells were transfected with plasmids encoding cyclin D3 and CDK4-HA in the absence or continuous presence of 1-NMPP1 for 48 h. Cell lysates were immunoprecipitated (IP) with anti-cyclin D3 or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunoblotting with anti-CDK4 antibody, which detected both ectopic and endogenous CDK4. (B) HCT116 K7AS cells were infected during 24 h by lentiviruses encoding Tet-On inducible wt or S178P CDK6-Flag with or without lentiviruses encoding Tet-On inducible cyclin D3. The expression of these proteins in the continuous presence (+) or absence (−) of 1-NMPP1 was induced by doxycycline 16 h prior to cell restimulation by fetal bovine serum (FBS) for 16 h. Western blotting detection of endogenous and ectopic (arrow) CDK6 from whole cell extracts is shown in the upper panel (NI, not infected). Cell lysates were immunoprecipitated (IP) with anti-Flag, anti-cyclin D3 or anti-p21 antibodies and separated by 2D gel electrophoresis followed by immunoblotting with anti-CDK6 antibody, which detected both ectopic and endogenous (e) CDK6. (C) T172 phosphorylation of endogenous CDK4 resists inhibition of CDK2 and CDK7 by roscovitine in p21-null (p21−/−) cells. Roscovitine (rosco; +) or vehicle (−) were administered for 16 h to asynchronously growing wild-type (WT) and p21−/− HCT116 cells cultured in parallel. Cell lysates were immunoprecipitated (IP) with anti-cyclin D1 (D1) or anti-cyclin D3 (D3) antibodies and separated by 2D gel electrophoresis followed by CDK4 immunodetection. Arrows, phosphorylated forms of CDK4 and CDK6-Flag; arrowheads in (B), unphosphorylated form of CDK6-Flag.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Transfection, Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Infection, Expressing, Phospho-proteomics, Inhibition, Cell Culture, Immunodetection

Journal: PLoS Genetics

Article Title: CDK4 T172 Phosphorylation Is Central in a CDK7-Dependent Bidirectional CDK4/CDK2 Interplay Mediated by p21 Phosphorylation at the Restriction Point

doi: 10.1371/journal.pgen.1003546

Figure Lengend Snippet: CDK7 inhibition reveals novel positive feedbacks mediated by S130 phosphorylation of p21, which is catalyzed by active CDK4 and CDK2 to permit T172 phosphorylation and activation of CDK4 complexes stabilized by p21 binding. S130 phosphorylation of p21 thus amplifies CDK4 activation and subsequently leads to increased degradation of p21, which in turn facilitates CDK2 activation. Those retrocontrols of CDK4 activation should contribute to explaining the irreversibility of the cell cycle commitment at the R point. Green/red colors indicate a final positive/negative influence on R point passage. 1 , 2 , two different levels of CDK7 action demonstrated by the present observations. CDK4 AK?, hypothetical CDK4-activating kinase(s) phosphorylating p21-unbound CDK4 and S178P CDK6 during CDK7 inhibition.

Article Snippet: (Co-)Immunoprecipitations were performed as described , using the following antibodies : monoclonal antibodies against cyclin D1 (DCS-11) or cyclin D3 (DCS-28) (all from Neomarkers); monoclonal antibody against HA epitope (F7) and polyclonal antibodies against p21 (C-19), CDK6 (C-21), CDK7 (C-19) and cyclin H (C-18) (all from Santa Cruz Biotechnology); monoclonal anti-Flag (M2) (Sigma).

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 1. Up-regulation of cyclin D2 occurs in parallel with down-regulation of p27 at the G0-G1 transition. (A) Serum-deprived NIH 3T3 cells, MEFs, or mouse primary lymphocytes were stimulated for the indicated times by exposure to medium containing 10% calf serum, 10% fetal bovine serum, or 10 nM phorbol 12,13-dibutyrate and 300 nM ionomycin, respectively. The percentage of cells in S phase was determined by flow cytometry. (B) Lysates prepared from the cells described above were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. HSP70 was examined as an internal control. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Cytometry, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 2. Depletion of cyclin D2 inhibits the cytoplasmic translocation of p27 in early G1 phase. (A) NIH 3T3 cells were infected with retroviral vectors encoding shRNAs specific for EGFP (control), cyclin D2 (D2-KD), or p27 (p27-KD) mRNAs or HA-tagged p27 and were arrested in G0 phase by serum deprivation for 72 h. The cells were then fixed immediately (0 h) or after stimulation for 4 h with 10% calf serum to induce synchronized progression of the cell cycle from G0 to early G1 phase. The cells were subjected to immunofluorescence analysis with antibodies to cyclin D2 and to p27 or HA and were stained with Hoechst 33258. Arrowheads indicate cytoplasmic signals of each protein. Arrows indicate nuclear accumulation of p27 in cells devoid of endogenous cyclin D2. Magnification, 40. (B) The cells depleted of cyclin D2 or p27 and stimulated for 4 h as described above were lysed and subjected to immunoblot (IB) analysis with antibodies to cyclin D2 or p27. (C) Quantitative analysis of the subcellular localization of p27 in cells depleted of cyclin D2 and control cells stimulated for 4 h as described above. A total of 65 to 120 cells was scored for each sample. Data represent the percentages of cells showing a predominantly nuclear localization of p27 and are means standard errors of the means from three independent experiments. (D) Cells expressing HA-p27 (or infected with the corresponding empty vector [Mock]) and stimulated for 4 h as described above were lysed and subjected to IB analysis with antibodies to p27. Bands corresponding to the exogenous (Exo.) and endogenous (Endo.) proteins are indicated.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Translocation Assay, Infection, Retroviral, Control, Staining, Western Blot, Expressing, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 3. Depletion of cyclin D2 delays p27 degradation at the G0-G1 transition and inhibits progression of the cell cycle. (A) NIH 3T3 cells were infected with retroviral vectors for EGFP (control), cyclin D1 (D1-KD), cyclin D2 (D2-KD), or cyclin D3 (D3-KD) shRNAs, arrested in G0 phase by serum deprivation, and stimulated by reexposure to 10% serum for the indicated times. Cell lysates were then subjected to immunoblot (IB) analysis with antibodies to p27 or to cyclin D1, D2, or D3. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. (B) NIH 3T3 cells were subjected to RNAi as described for panel A, arrested in G0 phase, lysed, and subjected to immunoprecipitation (IP) with antibodies to p27. The resulting precipitates as well as the original cell lysates (Input) were subjected to IB analysis with antibodies to cyclin D1, D2, or D3, to CDK4, or to p27. (C) Lysates of HEK293T cells expressing various amounts of HA-tagged p27 and similar amounts of M2-cyclin D1 or M2-cyclin D2 were subjected to IP with antibodies to HA (HA11). The resulting precipitates as well as the original cell lysates (Input) were subjected to IB analysis with antibodies to HA or to the Myc epitope. (D) NIH 3T3 cells were subjected to RNAi for EGFP or cyclin D2 followed by RNAi for EGFP or Skp2, arrested in G0 phase by serum deprivation for 72 h, and stimulated by reexposure to 10% calf serum for the indicated times. Cell lysates were then subjected to IB analysis with antibodies to p27 or to cyclin D2. Those cells which were growing in asynchronized conditions were also harvested, and the lysates from the cells were subjected to IB analysis with antibodies to HSP70 (control) and Skp2. (E) NIH 3T3 cells were subjected to RNAi as described for panel D, arrested in G0 phase by serum deprivation for 72 h, and harvested or stimulated by reexposure to 10% serum for 14 h. Cells were exposed to 10 M bromodeoxyuridine (BrdU) for 1 h before harvest. The cells were then subjected to fluorescence-activated cell sorter analysis with antibodies to BrdU. Data represent the percentages of cells positive for BrdU staining and are means standard errors of the means from two independent experiments.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Infection, Retroviral, Control, Western Blot, Immunoprecipitation, Expressing, BrdU Staining

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 4. Overexpression of cyclin D2 induces translocation of p27 from the nucleus to the cytoplasm. (A) NIH 3T3 cells were infected with a retroviral vector for M2-cyclin D1 or M2-cyclin D2 or with the corresponding empty vector (Mock) and were then arrested in G0 phase by serum deprivation for 72 h. The cells were fixed and subjected to immunofluorescence analysis with antibodies to the Myc epitope and to p27. (B) C2C12 cells were infected as described above, arrested in G0 phase by serum deprivation for 48 h, and subjected to immunofluorescence analysis as described above. (C) NIH 3T3 cells infected with the vector for M2-cyclin D2 or the empty vector as described above were arrested in G0 phase, after which cell lysates were prepared and subjected to immunoprecipitation (IP) with antibodies to p27 or to HA (Y-11). The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to CRM1, to the Myc epitope, or to p27. The abundance of exogenous cyclin D2 was relatively low intentionally because a high level of cyclin D2 expression results in a substantial reduction in the amount of p27 (see Fig. 5A), making it difficult to compare precisely the amounts of CRM1 bound to p27. (D) MEFs derived from p27S10A

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Over Expression, Translocation Assay, Infection, Retroviral, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 5. Overexpression of cyclin D2 promotes p27 degradation at the G0-G1 transition. (A) NIH 3T3 cells infected with a retroviral vector encoding M2-cyclin D1 or M2-cyclin D2 or with the corresponding empty vector were arrested in G0 phase by serum deprivation for 72 h and then reexposed to 10% calf serum for the indicated times. Cell lysates were subjected to immunoblot (IB) analysis with antibodies to p27, to cyclin D1, or to cyclin D2. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. The positions of bands corresponding to exogenous (Exo.) and endogenous (Endo.) proteins are shown. (B) C2C12 cells infected with retroviral vectors as described above were arrested in G0 phase by serum deprivation for 48 h and then reexposed to 10% fetal bovine serum for the indicated times. Cell lysates were subjected to IB analysis as described above. (C) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2 or with the empty vector were deprived of serum for 72 h, pulse-labeled with [35S]methionine and [35S]cysteine for 2 h, washed, and incubated for the indicated chase times in serum-deficient medium. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to p27, and the resulting precipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeled proteins were detected by autoradiography (top), and the intensity of the 35S-p27 bands was quantified and plotted relative to the values at time zero. Data are means from three independent experiments. (D) NIH 3T3 cells infected with retroviral vectors and arrested in G0 phase as described above were lysed and subjected to IP with antibodies to p27 or to

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Over Expression, Infection, Retroviral, Plasmid Preparation, Western Blot, Labeling, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 6. Cyclin D2 is phosphorylated on Thr280 in a manner independent of CDK4/6 activity. (A) Alignment of amino acid sequences in the COOH-terminal region of mouse cyclins D1 and D2. Conserved amino acids are boxed. The Thr286 phosphorylation site of cyclin D1 and the corresponding residue (Thr280) of cyclin D2 are indicated by the asterisk. (B) HEK293T cells expressing 3FLAG (F3)-tagged wild-type (WT) cyclin D2 or cyclin D2(T280A), or those transfected with the corresponding empty vector, were incubated for 4 h with 10 M MG132 (proteasome inhibitor) and exposed for the final 30 min of the incubation to 50 nM calyculin A and 5 M cyclosporine A (phosphatase inhibitors). Cell lysates were then subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to phosphothreonine (p-Thr). The original cell lysates (Input) were also subjected to IB analysis with antibodies to FLAG. (C) HEK293T cells expressing F3-tagged WT cyclin D2 and HA-tagged dnCDK4/6, as indicated, were incubated with MG132, calyculin A, and cyclosporine A as described for panel B. Cell lysates were then subjected to IP with antibodies to FLAG, and the resulting precipitates were subjected to IB analysis with antibodies to p-Thr. p-Thr was detected in dnCDK4/6 as well as in cyclin D2 (arrows). The original cell lysates (Input) were also subjected to IB analysis with antibodies to FLAG, to CDK4, and to CDK6. Exo., exogenous; Endo., endogenous. (D) NIH 3T3 cells infected with retroviral vectors for HA-tagged dnCDK4/6 or with the corresponding empty vector (Mock) were arrested in G0 phase, stimulated by exposure to 10% calf serum for 4 h to induce synchronized progression from G0 to early G1 phase, fixed, and subjected to immunofluorescence analysis with antibodies to cyclin D2 and to HA (Y-11). Magnification, 40.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Activity Assay, Phospho-proteomics, Residue, Expressing, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, Infection, Retroviral

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 7. Cyclin D2(T280A) inhibits nuclear export of p27 and delays its degradation. (A) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2(T280A) or with the corresponding empty vector (Mock) were arrested in G0 phase by serum deprivation for 72 h. Cell lysates were then subjected to immunoprecipitation (IP) with antibodies to p27. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to the Myc epitope, to CDK4, and to p27. (B) NIH 3T3 or C2C12 cells infected with a retroviral vector for M2-tagged wild-type (WT) cyclin D2 or cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation, fixed, and subjected to immunofluorescence analysis with antibodies to the Myc epitope and to p27. Magnification, 40. (C) NIH 3T3 cells infected with a retroviral vector for M2-tagged WT cyclin D2 or cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation and then reexposed to 10% serum for the indicated times. Cell lysates were subjected to IB analysis with antibodies to p27 or to cyclin D2. The positions of the exogenous (Exo.) and endogenous (Endo.) cyclin D2 proteins are indicated. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. (D) Quantitation of p27 band intensities relative to the corresponding values for time zero in experiments similar to the experiment with results shown in panel C. Data are means from three independent experiments. (E) An experiment similar to that with results shown in panel C was performed with C2C12 cells. (F) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation, reexposed to 10% serum for 3 h, and then incubated in the additional presence of cycloheximide (CH) (50 g/ml) for the indicated times. Cell lysates were subjected to IB analysis with antibodies to p27 or to the Myc epitope. The asterisk indicates nonspecific bands.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Infection, Retroviral, Plasmid Preparation, Immunoprecipitation, Western Blot, Quantitation Assay, Incubation

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: p16 INK4A forms intermolecular disulfides upon exposure to oxidants. (A) Analysis of Immuno-precipitated FLAG-p16 INK4A by non-reducing SDS-PAGE and Western Blot shows that part of the FLAG-p16 INK4A migrates at about double the molecular weight upon 5 min treatment with low amounts of the thiol-specific oxidant diamide (left panel) or H 2 O 2 (right panel). Reduction (R) prior to SDS-PAGE abolishes the shift in molecular weight, indicating that it is indeed due to an intermolecular disulfide (see also for confirmation that the high molecular weight form of p16 INK4A is an S–S-dependent homodimer). (B) S–S-dependent p16 INK4A homodimerization upon 200 μM diamide (left) or 200 μM H 2 O 2 (right) occurs rapidly, coincides with accumulation of p16 INK4A protein levels and oxidation as well as accumulation are fully dependent on C72. (C) Endogenous p16 INK4A and over-expressed FLAG-p16 INK4A accumulate in response to 200 μM diamide with similar kinetics. Note that p16 INK4A C72A does not accumulate whereas endogenous p16 INK4A does, suggesting that endogenous p16 INK4A levels are also regulated by Cys-oxidation. (IP: immunoprecipitation, WB: Western Blot). All Western blots shown in are typical results of several repeats (N ≥ 3 for all experiments).

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: SDS Page, Western Blot, Molecular Weight, High Molecular Weight, Immunoprecipitation

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: In vitro oxidation of p16 INK4A (A) 1 H 15 N HSQC solution NMR spectrum of recombinant p16 INK4A in the reduced state (blue) and after S-glutathionylation (magenta). Amino acids with large chemical shift changes are labeled. (B) Cartoon representation of the p16 INK4A structure. A color gradient from white (unaffected) to red (strongly affected) shows the influence of S-glutathionylation on the chemical shift. (C) The redox potential of C72 is 198.3 ± 1.7 mV, as determined from intensity changes of four well-separated amino acids by titration of the reduced protein with oxidized glutathione. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: In Vitro, Recombinant, Labeling, Titration

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: p16 INK4A forms amyloid fibrils under oxidizing conditions (A) 1 H, 15 N HSQC solution NMR spectra of recombinant p16 INK4A before (blue) and after (orange) oxidation with 50 mM H 2 O 2 for 10 h at room temperature (orange). The collapse of the peaks to the center of the spectrum suggests formation of unstructured or aggregated protein. (B) Negative-stained transmission electron micrographs of diamide-treated p16 INK4A showing the presence of amyloid fibrils. Right panel is at higher magnification, arrows highlight the twisted morphology which is typical of amyloid fibrils. (C) Fibril formation of p16 INK4A monitored by thioflavin-T fluorescence measurement. Error bars represent standard deviation from four measurements. (D) Fibril formation of p16 INK4A monitored by Congo red absorption. The absorption maximum shifts when bound to oxidized p16 INK4A , indicating the presence of amyloid fibrils. (E) Amide I region of the attenuated-total-reflectance Fourier-transform infrared spectra of p16 INK4A monomer and fibrils. Peak maxima indicate that the structure contains primarily α-helix for monomers and cross-β sheet for fibrils. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: Recombinant, Staining, Transmission Assay, Fluorescence, Standard Deviation, Fourier Transform Infrared Spectroscopy

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: C72 is important for fibrillar morphology and ThT binding. (A) Negative-stain transmission electron micrographs of p16 INK4A treated with 200 μM hydrogen peroxide (left) and 200 μM diamide (right). Top: WT p16 INK4A . Middle: p16 INK4A -C72S. Bottom: GS-p16 INKA . (B) Thioflavin-T fluorescence kinetics assay of p16 INK4A samples (WT, C72S and GS) with 200 μM diamide addition (top) 200 μM H 2 O 2 addition (middle) and without oxidizing agents (bottom). Three replicates were measured per sample and each are separately plotted.

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: Binding Assay, Staining, Transmission Assay, Fluorescence

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: p16 INK4A aggregates in live cells in response to oxidation . (A) Set up for the Filter Trap assay for the detection of aggregates in cell lysates. (B) Results for the Filter Trap assay. Note that the majority of p16 INK4A in the pellet is in the form of S-S-linked homodimers after lysis. p16 INK4A , but not p16 INK4A C72A, was trapped on the filter membrane upon treatment with 200 μM diamide and trapping was prevented by pretreatment with DTT. Equal amounts of p16 INK4A and p16 INK4A C72A were used as input for the filter trap assay. Note that the input for the Filter Trap assay runs mainly as a S–S-dependent dimer after boiling in non-reducing SDS-PAGE sample buffer. R: reducing, NR: non-reducing. (C) Aggregates of p16 INK4A peak around 30 min and are then cleared over time. (D) Endogenous p16 INK4A also forms S–S-linked dimers that (partially) form aggregates in the insoluble pellet fraction. Note that also mixed S–S-linked dimers of endogenous and overexpressed p16INK4A can be observed in lanes where FLAG-p16INK4A was transfected. n.i.: IP with non-immune serum as a control. p16(r): rabbit anti-p16INK4A, p16(m): mouse anti-p16INK4A, e.v.: empty vector, Red: reducing, Non-Red: non-reducing. All experiments were performed at least 3 times. Representative experiments are shown.

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: TRAP Assay, Lysis, Membrane, SDS Page, Transfection, Control, Plasmid Preparation

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: Oxidation of p16 INK4A impairs its inhibitory function towards CDK4/6. (A) In vitro CDK4/6 kinase assay on WT p16 INK4A pulldowns shows that the oxidation of p16 INK4A -C72 impairs its inhibitory function. Note that oxidation or mutation of Cys72 does not affect the amount of CDK4/6 that is co-immunoprecipitated. (R: Reducing, NR: Non-reducing). All Western blots shown in are typical results of several repeats (N ≥ 3 for all experiments). (B) qPCR analysis showing the expression of WT p16 INK4A WT or p16 INK4A -C72A (top) in COLO-829 cells upon induction of expression from the respective pINDUCER20-p16 INK4A construct. CCNE2 expression (bottom) is strongly repressed both by WT p16 INK4A and p16 INK4A -C72A. Diamide treatment relieves this repression only in the WT p16 INK4 expressing cell line. Note that the inducible system is somewhat leaky, which could explain induction of CCNE2 in the p16 INK4A WT cells by diamide in the absence of doxycycline. The mean plus SEM is displayed from n = 5 biological replicates. *p < 0.05, **p < 0.01, pairwise comparison using a two-sided t -test comparing cells carrying the inducible WT p16 INK4 or p16 INK4A -C72A construct in each condition.

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques: In Vitro, Kinase Assay, Mutagenesis, Immunoprecipitation, Western Blot, Expressing, Construct, Comparison

Journal: Redox Biology

Article Title: Cysteine oxidation triggers amyloid fibril formation of the tumor suppressor p16 INK4A

doi: 10.1016/j.redox.2019.101316

Figure Lengend Snippet: Evolutionary analysis of p16 INK4A and its paralogue p15 INK4B . (A) Conservation of Cys72 in human p16 INK4A and (B) Cys74 in its paralogue p15 INK4B throughout vertebrate evolution. Note that ENSEMBL assigns some non-mammalian orthologues to either p16 INK4A or p15 INK4B whereas gene duplication to yield these paralogues did not take place until mammalian species had branched off . Sequences are named by ENSEMBL ID . The human orthologues are indicated with * and in bold. Cysteines conserved at the position homologous to human p16 INK4A Cys72 are indicated on yellow background, other cysteines are in yellow text. The darker the purple shading the more conserved the amino acids. Note that several distant branches of mammals have lost the cysteine homologous to human p16 INK4A Cys72. See for a schematic tree of vertebrate and mammalian evolution for reference. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The following antibodies were used in this study: FLAG (M2 F3165 Sigma Aldrich), p16 INK4A (10883 Proteintech), p16 INK4A (ab16123 Abcam), CDK4 (C-22 Santa Cruz), CDK6 (C-21 Santa Cruz), tubulin (CP06 Merck Millipore), Phospho-Rb Ser807/811 (9308 Cell Signaling).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 1. Up-regulation of cyclin D2 occurs in parallel with down-regulation of p27 at the G0-G1 transition. (A) Serum-deprived NIH 3T3 cells, MEFs, or mouse primary lymphocytes were stimulated for the indicated times by exposure to medium containing 10% calf serum, 10% fetal bovine serum, or 10 nM phorbol 12,13-dibutyrate and 300 nM ionomycin, respectively. The percentage of cells in S phase was determined by flow cytometry. (B) Lysates prepared from the cells described above were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. HSP70 was examined as an internal control. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Cytometry, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 2. Depletion of cyclin D2 inhibits the cytoplasmic translocation of p27 in early G1 phase. (A) NIH 3T3 cells were infected with retroviral vectors encoding shRNAs specific for EGFP (control), cyclin D2 (D2-KD), or p27 (p27-KD) mRNAs or HA-tagged p27 and were arrested in G0 phase by serum deprivation for 72 h. The cells were then fixed immediately (0 h) or after stimulation for 4 h with 10% calf serum to induce synchronized progression of the cell cycle from G0 to early G1 phase. The cells were subjected to immunofluorescence analysis with antibodies to cyclin D2 and to p27 or HA and were stained with Hoechst 33258. Arrowheads indicate cytoplasmic signals of each protein. Arrows indicate nuclear accumulation of p27 in cells devoid of endogenous cyclin D2. Magnification, 40. (B) The cells depleted of cyclin D2 or p27 and stimulated for 4 h as described above were lysed and subjected to immunoblot (IB) analysis with antibodies to cyclin D2 or p27. (C) Quantitative analysis of the subcellular localization of p27 in cells depleted of cyclin D2 and control cells stimulated for 4 h as described above. A total of 65 to 120 cells was scored for each sample. Data represent the percentages of cells showing a predominantly nuclear localization of p27 and are means standard errors of the means from three independent experiments. (D) Cells expressing HA-p27 (or infected with the corresponding empty vector [Mock]) and stimulated for 4 h as described above were lysed and subjected to IB analysis with antibodies to p27. Bands corresponding to the exogenous (Exo.) and endogenous (Endo.) proteins are indicated.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Translocation Assay, Infection, Retroviral, Control, Staining, Western Blot, Expressing, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 3. Depletion of cyclin D2 delays p27 degradation at the G0-G1 transition and inhibits progression of the cell cycle. (A) NIH 3T3 cells were infected with retroviral vectors for EGFP (control), cyclin D1 (D1-KD), cyclin D2 (D2-KD), or cyclin D3 (D3-KD) shRNAs, arrested in G0 phase by serum deprivation, and stimulated by reexposure to 10% serum for the indicated times. Cell lysates were then subjected to immunoblot (IB) analysis with antibodies to p27 or to cyclin D1, D2, or D3. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. (B) NIH 3T3 cells were subjected to RNAi as described for panel A, arrested in G0 phase, lysed, and subjected to immunoprecipitation (IP) with antibodies to p27. The resulting precipitates as well as the original cell lysates (Input) were subjected to IB analysis with antibodies to cyclin D1, D2, or D3, to CDK4, or to p27. (C) Lysates of HEK293T cells expressing various amounts of HA-tagged p27 and similar amounts of M2-cyclin D1 or M2-cyclin D2 were subjected to IP with antibodies to HA (HA11). The resulting precipitates as well as the original cell lysates (Input) were subjected to IB analysis with antibodies to HA or to the Myc epitope. (D) NIH 3T3 cells were subjected to RNAi for EGFP or cyclin D2 followed by RNAi for EGFP or Skp2, arrested in G0 phase by serum deprivation for 72 h, and stimulated by reexposure to 10% calf serum for the indicated times. Cell lysates were then subjected to IB analysis with antibodies to p27 or to cyclin D2. Those cells which were growing in asynchronized conditions were also harvested, and the lysates from the cells were subjected to IB analysis with antibodies to HSP70 (control) and Skp2. (E) NIH 3T3 cells were subjected to RNAi as described for panel D, arrested in G0 phase by serum deprivation for 72 h, and harvested or stimulated by reexposure to 10% serum for 14 h. Cells were exposed to 10 M bromodeoxyuridine (BrdU) for 1 h before harvest. The cells were then subjected to fluorescence-activated cell sorter analysis with antibodies to BrdU. Data represent the percentages of cells positive for BrdU staining and are means standard errors of the means from two independent experiments.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Infection, Retroviral, Control, Western Blot, Immunoprecipitation, Expressing, BrdU Staining

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 4. Overexpression of cyclin D2 induces translocation of p27 from the nucleus to the cytoplasm. (A) NIH 3T3 cells were infected with a retroviral vector for M2-cyclin D1 or M2-cyclin D2 or with the corresponding empty vector (Mock) and were then arrested in G0 phase by serum deprivation for 72 h. The cells were fixed and subjected to immunofluorescence analysis with antibodies to the Myc epitope and to p27. (B) C2C12 cells were infected as described above, arrested in G0 phase by serum deprivation for 48 h, and subjected to immunofluorescence analysis as described above. (C) NIH 3T3 cells infected with the vector for M2-cyclin D2 or the empty vector as described above were arrested in G0 phase, after which cell lysates were prepared and subjected to immunoprecipitation (IP) with antibodies to p27 or to HA (Y-11). The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to CRM1, to the Myc epitope, or to p27. The abundance of exogenous cyclin D2 was relatively low intentionally because a high level of cyclin D2 expression results in a substantial reduction in the amount of p27 (see Fig. 5A), making it difficult to compare precisely the amounts of CRM1 bound to p27. (D) MEFs derived from p27S10A

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Over Expression, Translocation Assay, Infection, Retroviral, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 5. Overexpression of cyclin D2 promotes p27 degradation at the G0-G1 transition. (A) NIH 3T3 cells infected with a retroviral vector encoding M2-cyclin D1 or M2-cyclin D2 or with the corresponding empty vector were arrested in G0 phase by serum deprivation for 72 h and then reexposed to 10% calf serum for the indicated times. Cell lysates were subjected to immunoblot (IB) analysis with antibodies to p27, to cyclin D1, or to cyclin D2. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. The positions of bands corresponding to exogenous (Exo.) and endogenous (Endo.) proteins are shown. (B) C2C12 cells infected with retroviral vectors as described above were arrested in G0 phase by serum deprivation for 48 h and then reexposed to 10% fetal bovine serum for the indicated times. Cell lysates were subjected to IB analysis as described above. (C) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2 or with the empty vector were deprived of serum for 72 h, pulse-labeled with [35S]methionine and [35S]cysteine for 2 h, washed, and incubated for the indicated chase times in serum-deficient medium. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to p27, and the resulting precipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeled proteins were detected by autoradiography (top), and the intensity of the 35S-p27 bands was quantified and plotted relative to the values at time zero. Data are means from three independent experiments. (D) NIH 3T3 cells infected with retroviral vectors and arrested in G0 phase as described above were lysed and subjected to IP with antibodies to p27 or to

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Over Expression, Infection, Retroviral, Plasmid Preparation, Western Blot, Labeling, Incubation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 6. Cyclin D2 is phosphorylated on Thr280 in a manner independent of CDK4/6 activity. (A) Alignment of amino acid sequences in the COOH-terminal region of mouse cyclins D1 and D2. Conserved amino acids are boxed. The Thr286 phosphorylation site of cyclin D1 and the corresponding residue (Thr280) of cyclin D2 are indicated by the asterisk. (B) HEK293T cells expressing 3FLAG (F3)-tagged wild-type (WT) cyclin D2 or cyclin D2(T280A), or those transfected with the corresponding empty vector, were incubated for 4 h with 10 M MG132 (proteasome inhibitor) and exposed for the final 30 min of the incubation to 50 nM calyculin A and 5 M cyclosporine A (phosphatase inhibitors). Cell lysates were then subjected to immunoprecipitation (IP) with antibodies to FLAG, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to phosphothreonine (p-Thr). The original cell lysates (Input) were also subjected to IB analysis with antibodies to FLAG. (C) HEK293T cells expressing F3-tagged WT cyclin D2 and HA-tagged dnCDK4/6, as indicated, were incubated with MG132, calyculin A, and cyclosporine A as described for panel B. Cell lysates were then subjected to IP with antibodies to FLAG, and the resulting precipitates were subjected to IB analysis with antibodies to p-Thr. p-Thr was detected in dnCDK4/6 as well as in cyclin D2 (arrows). The original cell lysates (Input) were also subjected to IB analysis with antibodies to FLAG, to CDK4, and to CDK6. Exo., exogenous; Endo., endogenous. (D) NIH 3T3 cells infected with retroviral vectors for HA-tagged dnCDK4/6 or with the corresponding empty vector (Mock) were arrested in G0 phase, stimulated by exposure to 10% calf serum for 4 h to induce synchronized progression from G0 to early G1 phase, fixed, and subjected to immunofluorescence analysis with antibodies to cyclin D2 and to HA (Y-11). Magnification, 40.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Activity Assay, Phospho-proteomics, Residue, Expressing, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, Infection, Retroviral

Journal: Molecular and Cellular Biology

Article Title: Cyclin D2 Translocates p27 out of the Nucleus and Promotes Its Degradation at the G 0 -G 1 Transition

doi: 10.1128/mcb.00862-06

Figure Lengend Snippet: FIG. 7. Cyclin D2(T280A) inhibits nuclear export of p27 and delays its degradation. (A) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2(T280A) or with the corresponding empty vector (Mock) were arrested in G0 phase by serum deprivation for 72 h. Cell lysates were then subjected to immunoprecipitation (IP) with antibodies to p27. The resulting precipitates as well as the original cell lysates (Input) were subjected to immunoblot (IB) analysis with antibodies to the Myc epitope, to CDK4, and to p27. (B) NIH 3T3 or C2C12 cells infected with a retroviral vector for M2-tagged wild-type (WT) cyclin D2 or cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation, fixed, and subjected to immunofluorescence analysis with antibodies to the Myc epitope and to p27. Magnification, 40. (C) NIH 3T3 cells infected with a retroviral vector for M2-tagged WT cyclin D2 or cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation and then reexposed to 10% serum for the indicated times. Cell lysates were subjected to IB analysis with antibodies to p27 or to cyclin D2. The positions of the exogenous (Exo.) and endogenous (Endo.) cyclin D2 proteins are indicated. The band indicated by the asterisk is attributable to cross-reaction of the antibodies to cyclin D2 with cyclin D1. (D) Quantitation of p27 band intensities relative to the corresponding values for time zero in experiments similar to the experiment with results shown in panel C. Data are means from three independent experiments. (E) An experiment similar to that with results shown in panel C was performed with C2C12 cells. (F) NIH 3T3 cells infected with a retroviral vector for M2-cyclin D2(T280A) or with the corresponding empty vector were arrested in G0 phase by serum deprivation, reexposed to 10% serum for 3 h, and then incubated in the additional presence of cycloheximide (CH) (50 g/ml) for the indicated times. Cell lysates were subjected to IB analysis with antibodies to p27 or to the Myc epitope. The asterisk indicates nonspecific bands.

Article Snippet: Immunoblots were probed with antibodies to HA (HA11), to FLAG (M2), to the Myc epitope (9E10), to p27 (Transduction Laboratories), to cyclin D1 (72-13G [Santa Cruz Biotechnology]), to cyclin D2 (M-20 [Santa Cruz Biotechnology]), to cyclin D3 (Transduction Laboratories), to CDK4 (DCS-31 [Sigma] or C-22 [Santa Cruz Biotechnology]), to CDK6 (C-21 [Santa Cruz Biotechnology]), to human pRb phosphorylated on Ser780 (MBL), to phosphothreonine (Cell Signaling), to HSP70 (Transduction Laboratories), to CRM1 (Transduction Laboratories), or to KPC1 or KPC2 (33).

Techniques: Infection, Retroviral, Plasmid Preparation, Immunoprecipitation, Western Blot, Quantitation Assay, Incubation

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Experimental design of the loss‐of‐function screening. “ n ” indicates the number of biological replicates of the screening (each performed in triplicate). Significance ( P ) was calculated by two‐sided, unpaired t ‐test. Cell viability of MDAH cells transduced with the indicated shRNAs and treated with 140 μg/ml of CBDCA for 16 h. The corresponding cell lysates were analyzed by Western blot. Data represent the mean ± SD of two independent experiments each performed in duplicate (two‐sided, unpaired t ‐test). CDDP and CBDCA dose–response curve of MDAH cells, silenced or not for CDK6. Results are expressed as percentage of viable cells with respect to untreated cells and the resulted IC50 (half maximal inhibitory concentrations) as the mean value of three independent experiments ± SD each performed in triplicate. Colony assay and its quantification performed on MDAH cells transduced as indicated and treated or not with CDDP (0.3 μg/ml) for 72 h and then released for 72 h. Data represent the mean ± SD of two independent experiments performed in triplicate (two‐sided, unpaired t ‐test). Growth curve of control or CDK6 stably silenced MDAH cells. The corresponding cell lysate was analyzed by Western blot. Data represent the mean ± SD of two independent experiments (each performed in triplicate). Western blot analysis of PARP1 cleavage and RB1, pRB1 S780 , and CDK6 expressions in MDAH cells transduced as indicated and treated with vehicle (V) or with CBDCA, as in (B), and then released (R) for 8 and 24 h. Cell cycle distribution of MDAH cells transduced with control or CDK6‐specific shRNAs, evaluated by flow cytometry 72 h post‐transduction. The corresponding cell lysates were analyzed by Western blot. SA‐β‐Galactosidase‐positive cells/field in MDAH cells transduced as indicated and stained 72 and 96 h post‐transduction (left) or silenced and then treated with CBDCA, as in (B) (right). Data represent the mean ± SD of two independent experiments, each performed in triplicate (two‐sided, unpaired t ‐test). Typical fields of stained cells are shown in the inset. Arrows point to β‐galactosidase‐positive cells. Western blot analysis of the expression and cleavage of PARP1 and caspase‐3 and the expression of γH2AX and CDK6, in MDAH cells silenced using control or CDK6‐specific shRNAs and treated with vehicle (V) or with CBDCA as in (B) (P) and released (R) for the indicated times. Immunofluorescence analysis evaluating the expression of γH2AX (red) in MDAH cells treated as indicated in (I). Nuclei were stained with propidium iodide (pseudocolored in blue). Release indicates cells treated with CBDCA as in (B) and allowed to repair for 8 h. Vinculin and tubulin were used as loading controls. Data information: In each panel, significant differences are evidenced by asterisks (** P < 0.01, *** P < 0.001, **** P < 0.0001), and the exact P ‐values of (B, D and H) are reported in Appendix Table S4 . Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam).

Techniques: Transduction, Western Blot, Colony Assay, Control, Stable Transfection, Flow Cytometry, Staining, Expressing, Immunofluorescence

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Table summarizing CDDP and CBDCA IC50 of OVCAR8 cells transduced with ctrl or CDK6‐specific shRNAs. Results are expressed as percentage of viable cells with respect to untreated cells and the resulting IC50 (half maximal inhibitory concentrations) are reported ( n = 3 performed in triplicate). Growth curve of control or CDK6 silenced OVCAR8 cells. Data represent the mean ± SD of two biological replicates (each performed in triplicate). The corresponding cell lysates were analyzed by Western blot for CDK6 expression. Vinculin was used as loading control Cell cycle distribution of OVCAR8 cells transduced with control or CDK6‐specific shRNAs evaluated by flow cytometry 72 h post‐transduction. The corresponding cell lysates were analyzed by Western blot for CDK6 expression. Tubulin was used as loading control. Western blot evaluating PARP expression and cleavage and γH2AX S139 and CDK6 expression in OVCAR8 cells transduced with control or CDK6 shRNA and treated with 60 μg/ml of CBDCA for 16 h (P) and the released in platinum‐free medium for the indicated times. V indicates control cells treated with vehicle. Tubulin was used as loading control. Cell viability of OVCAR8 cells transfected with CDK6 WT or empty vector and treated with increasing doses of CDDP for 16 h. Results are expressed as percentage of viable cells with respect to untreated cells. Data represent the mean ± SD of three biological replicates (two‐sided, unpaired t ‐test). The corresponding cell lysates were analyzed by Western blot for CDK6 expression. GRB2 was used as loading control. Data information: In each panel, significant differences are evidenced by asterisks (* P < 0.05, *** P < 0.001) and the exact P ‐values of (E) are reported in Appendix Table S4 . Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam).

Techniques: Transduction, Control, Western Blot, Expressing, Flow Cytometry, shRNA, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Schematic design of in vivo experiments with SKOV3ip xenografts testing the efficacy of suboptimal doses of CBDCA (20 mg/kg) and PD (150 mg/kg) alone and in combination. Analysis of tumor growth in each experimental group described in (A). Red arrows indicate CBDCA treatments; blue arrows indicate PD treatment (two‐sided, unpaired t ‐test). Error bars represent SD. Scattered dot plot reporting the weight of explanted tumors. Each dot represents one tumor. Bars indicate mean ± 95% CI (Mann–Whitney test). Representative image of explanted tumors is shown on the top. Western blot analysis of pRB1 S780 and Ki67 expression in tumors explanted from mice treated as described in (A). Numbers on the top indicate the analyzed tumors. GAPDH was used as loading control. Immunofluorescence analysis of γH2AX (red) in tumors explanted from mice treated as described in (A). Quantification of γH2AX in explanted tumors ( n = 3/group in which at least three randomly selected fields were studied) treated as indicated and evaluated as mean fluorescence per cell. Bars indicate mean ± 95% CI (Mann–Whitney test). Schematic design of in vivo experiments with MDAH transduced with sh‐ctrl (right flank) or sh‐CDK6 (left flank) and then subcutaneously injected in nude mice ( n = 6 mice for each group). Analysis of explanted tumor weight is reported in the scattered dot plot. Bars indicate mean ± 95% CI. Western blot reports the expression and phosphorylation of H2AX and RB1 in tumors explanted from a representative mouse (Mann–Whitney test). Quantification of RB1 (upper graph) and H2AX (lower graph) phosphorylation in tumors explanted from four different mice treated as described in (G). Vinculin was used as loading control. Data information: In each panel, significant differences are evidenced by asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001) and the exact P ‐values of (C, F, and H) are reported in Appendix Table S4 . Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam).

Techniques: In Vivo, MANN-WHITNEY, Western Blot, Expressing, Control, Immunofluorescence, Fluorescence, Transduction, Injection, Phospho-proteomics

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Table reporting CDK6‐specific phosphorylation targets ordered by their PR score [adapted from Anders et al ]. The PR score represents the normalized phosphorylation levels of the indicated protein with respect to RB used as positive control (PR = 100). Experimental design of the loss‐of‐function screening performed on MDAH cells to evaluate the effect of silencing CDK6 phosphorylation targets. “ n ” indicates the number of independent experiments performed. Significance ( P ) was calculated by two‐sided, unpaired t ‐test. Graph reporting the results of the loss‐of‐function screening described in (B). Data represent the mean ± SD of three independent experiments each performed in triplicate and graphed as survival ratio between of CBDCA‐treated (140 μg/ml for 16 h) and untreated cells. Significance was calculated using two‐sided, unpaired t ‐test. Significant differences are evidenced by asterisks (** P < 0.01), and the exact P ‐values reported in Appendix Table S4 . Western blot analysis reporting the expression of FOXO3 in the indicated EOC cell lines. Actin was used as loading control. Co‐IP analysis of endogenous CDK4 with FOXO3 and cyclin D1 (upper left panel) in MDAH cells treated with CDDP 7.5 or 15 μg/ml for the indicated times. In the right panels, expression of the same proteins in the corresponding lysates (Input) is reported. In the lower left panels are reported the Co‐IP analyses of cyclin D1 and cyclin D3 with FOXO3 and CDK6 in cells treated for 3 or 6 h with 15 μg/ml of CDDP. In the right lower panels is reported the expression of the same proteins in the corresponding lysates (Input). Tubulin was used as loading control. Dose–response curve on MDAH cells transduced with control, cyclin D1, or cyclin D3 shRNAs and treated with increasing doses of CDDP for 16 h (three biological replicates). The resulted CDDP IC50 is reported in the table. Lower panels show the Western blot analyses of the expression of cyclins D1 and D3 in the transduced cells. Vinculin was used as loading control. Error bars represent SD. Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam).

Techniques: Phospho-proteomics, Positive Control, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay, Transduction

Journal: EMBO Molecular Medicine

Article Title: CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

doi: 10.15252/emmm.201607012

Figure Lengend Snippet: Expression and phosphorylation of the indicated proteins, in MDAH cells silenced for CDK6 or FOXO3 treated with CDDP (15 μg/ml, 3 h) and released (R) for 3 or 16 h. Graph reports the normalized quantification of FOXO3 and CDK6, obtained by densitometric scanning of the blot (FOXO3/vinculin ratio). Data represent the mean ± SD of three independent experiments. Significance ( P ) was calculated by two‐sided, unpaired t ‐test. Expression of ATR in MDAH cells silenced for CDK6 and then transfected with FOXO3 WT , as indicated, and treated with CDDP as in (A). Expression of ATR, FOXO3, and CDK6 in MDAH cells treated with CDDP (15 μg/ml, 3 h) and PD (8 μM, 24 h), alone or in combination. ATR expression in tumors explanted from mice treated as indicated and described in Fig A. The number on top indicates the analyzed tumors. Enrichment of ATR promoter in FLAG‐IPs in chromatin immunoprecipitation (ChIP) analysis using cells transfected with empty vector (E.V.) or FLAG‐FOXO3. Data represent the mean ± SD of two independent experiments, each performed in duplicate. Data are expressed as fold enrichment with respect to control IgG. Enrichment of ATR promoter in ChIP analysis, using an anti‐FOXO3 or control‐IgG antibodies in cells treated with vehicle (V) or with CDDP (15 μg/ml, 3 h) (P) and then released (R) for 3 h as in (A). Data represent the mean of three biological replicates. Data are expressed as fold enrichment with respect to control IgG. Expression and phosphorylation of CHK1 in control and ATR‐silenced MDAH cells treated with CDDP as in (F). Typical images of normal mitosis and premature chromosome condensation (PCC), observed in MDAH cells transduced as indicated and then treated with CDDP and then allowed to recover for 6 h. Scale bar = 10 μm. Percentage of PCC (with respect to total nuclei) in MDAH cells treated with CDDP (15 μg/ml, 3 h) and then released for 6 h (R 6 h) in platinum‐free medium. V = vehicle. Table reporting the number of normal mitosis, PCC, and total nuclei counted in cells treated as in (I). Data information: Vinculin and tubulin were used as loading controls. In each panel, significant differences are evidenced by asterisks (* P < 0.05, ** P < 0.01) and the exact P ‐values of (A) is reported in Appendix Table S4 . Source data are available online for this figure.

Article Snippet: Primary antibodies used were as follows: vinculin (N‐19, 1:1,000), CDK6 (C‐21, sc‐177, 1:800), CDK4 (C‐22, sc‐260, 1:400), pRB1S780 (sc‐12901, 1:400), PSF (39‐1, sc‐101137, 1:800), CHK1 (G‐4, sc‐8408, 1:500), luciferase (sc‐32896), lamin A (C‐20 sc‐6214) (Santa Cruz Biotechnology); FOXO3 (75D8, #2497, 1:600), cyclin D3 (DCS22, #2936, 1:500), pCHK1S296 (#2349, 1:500), H2AX (D17A3, #7631, 1:500), ATR (#2790, 1:500), ATM (#2873, 1:500), and cleaved caspase‐3 Asp175 (#9661, 1:500) (Cell Signaling); cyclin D1 (DCS‐6, CC12, 1:1,000) (Millipore); γH2AXS139 (#2577, 1:500) (Upstate Biotechnology); RB1 (554136, 1:500) and GRB2 (610111, 1:300) (BD Biosciences); actin (A5060, 1:500), tubulin (T5168, 1:1,000), Flag (F3040), OP18 (O0138, 1:1,000), and V5 (A7345)‐ and HA (A2095)‐agarose conjugated (Sigma‐Aldrich Co); GFP (11 814 440 001, 1:500) (Roche); Ki67 (ab15580, 1:1,000) (Abcam).

Techniques: Expressing, Phospho-proteomics, Transfection, Chromatin Immunoprecipitation, Plasmid Preparation, Control