cyclin d1 Search Results


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  • 99
    Thermo Fisher cyclin d1
    Down-regulation of STAT3 regulates the expression of apoptotic and cell cycle related proteins in ECA109 cells. a <t>Cyclin</t> D1, c-Myc, Caspase-3, cleaved Caspase-3 proteins were analyzed by Western blotting in ECA109 cells after transfection with pSi-Scramble and pSi-STAT3. GAPDH was used as loading control. b Quantitative levels of aforementioned proteins against GAPDH. All data are represented as the mean ± SE, *P
    Cyclin D1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cyclin d1
    Expression of apoptosis-associated genes and cell cycle-associated genes in NB4 cells treated with different concentrations of sorafenib for 48 h. (A) Semi-quantitative polymerase chain reaction and (B) western blot analysis demonstrated that sorafenib increased the expression of caspase-8 and caspase-3, and decreased the expression of MCL1 and <t>cyclin</t> D1. *P
    Cyclin D1, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 926 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology cyclin d1
    <t>Cyclin</t> D1 does not accumulate in Fbxo4 −/− MEFs. (A) Growth curves for Fbxo4 +/+ and Fbxo4 −/− MEFs. Data are means ± SD of values obtained from three MEF preparations of each genotype. (B) Immunoblot analysis of cyclin
    Cyclin D1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 10420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cyclin d1
    Effects of downregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, <t>cyclin</t> A2, p21 and <t>cyclin</t> D1 protein expression in each group of SGC-7901 cells. (B) Comparison of protein levels in each group of SGC-7901 cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of SGC-7901 cells. (D) Comparison of protein levels in each group of SGC-7901 cells. **P
    Cyclin D1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam cyclin d1
    Effects of downregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, <t>cyclin</t> A2, p21 and <t>cyclin</t> D1 protein expression in each group of SGC-7901 cells. (B) Comparison of protein levels in each group of SGC-7901 cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of SGC-7901 cells. (D) Comparison of protein levels in each group of SGC-7901 cells. **P
    Cyclin D1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2915 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti cyclin d1
    <t>Cyclin</t> D1-CDK4 regulates gluconeogenesis in primary hepatocytes and in whole animals. a) Western-blot analysis of endogenous, forskolin-induced (Fsk) or adenovirally overexpressed (O/E) PGC-1α. Nuclear extracts of primary hepatocytes were used to immunoprecipitate PGC-1α. Cells were infected with GFP or PGC-1α 48hr prior to harvest. 10μM forskolin was added for 2hr before harvest. b) PD 0332991 increases forskolin-induced gluconeogenic gene expression. Primary hepatocytes were treated with 10μM forskolin for 1.5hr following 3hr of starvation medium incubation while 1μM PD 0332991 was added overnight (one-way ANOVA with Tukey post test, n=3). c) CDK4 knockdown increases forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). d) <t>Cyclin</t> D1 wild-type, but not cyclin D1 K112E mutant, suppresses forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). e) Phosphorylation of GCN5, FoxO1 N-terminus, FoxO1 C-terminus, FoxO3A and PGC-1α SR domain by cyclin D1-CDK4. f) PGC-1α knockdown blocks the increase of forskolin-induced gluconeogenic genes by fascaplysin in HepG2 cells. PGC-1α knockdown or a negative control HepG2 cells were treated with 30μM forskolin and 1μM fascaplysin overnight (one-way ANOVA with Tukey post test, n=3). g) GCN5 knockdown blunts the increase of gluconeogenic gene expression caused by CDK4 knockdown. qRT-PCR analysis of Pck1 and Gcn5 and western-blot of CDK4 and GCN5 knockdown are shown. All cells were infected with PGC-1α adenoviruses (one-way ANOVA with Tukey post test, n=15). h) PD 0332991 increases gluconeogenic genes when combined with GCN5 wild-type (WT) overexpression, but not with GCN5 T272A/S372A (AA) mutant. GFP infected cells shown as a comparison to GCN5 overexpressing cells. All cells were infected with PGC-1α adenoviruses (two-tailed unpaired t-test, n=6). I-j) Insulin levels measured from serum and western-blot analysis of Rb and AKT using nuclear (N) and cytoplasmic (C) liver extracts from mice treated with vehicle or 150mg/kg PD 0332991, shown in Fig. 2lm ( i : two-tailed unpaired t-test, n=18/GFP, n=17/PD 0332991). k) Levels of cyclin D1 and Rb phosphorylation in GFP or Cyclin D1 T286A tail-vein injected mice, shown in Fig. 2n-o . Statistical significance is represented by asterisk corresponding to * P
    Anti Cyclin D1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 2313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti cyclin d1
    Isoliquiritigenin regulates the members of PI3K/Akt/mTOR pathway in Ishikawa and ES-2 cells. ILQ treatment significantly reduced the expression level of p-Akt, p-mTOR, P70/S6K, and <t>Cyclin</t> D1 in both Ishikawa and ES-2 cells. * P
    Anti Cyclin D1, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1000 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc cyclind1
    Met protects HaCaT cells against RAGE overexpression-induced cell cycle arrest. After infected with pLV-IRES-eGFP-RAGE for 48 h, HaCaT cells were treated with Met at 100 μM for 48 h. Cell cycles were measured by PI staining and flow cytometry (A). Expression of cell cycle-related proteins, including p21, Gadd45a, CyclinB1, <t>CyclinD1,</t> and CDK4, was measured by Western blot (B). # P
    Cyclind1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novocastra cyclin d1
    Western blot analysis for Stat 3, Stat 3P, <t>cyclin</t> D1, and anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2 in human MM cell lines. Each lane contains 60 μg of protein extract from the following cell lines: lane 1 , Granta 519; lane 2 , KMS-20; lane 3 , U266; lane 4 , KMS-18; lane 5 , KMS-5; lane 6 , KMS-11; lane 7 , KMM1; lane 8 , KMS-12; lane 9 , OPM2. Lane 1 (Granta 519, mantle cell lymphoma cell line) and lane 8 (KMS12) represent the t(11;14) translocated cell lines. Expression of <t>cyclin</t> D1 is limited to these two cell lines. Lane 3 (U266) represents the cell line with known constitutive activation of Stat 3. Western blot analysis with the N-terminal anti-Stat 3 antibody demonstrates both Stat 3α and Stat 3β (92 and 83 kd, respectively) in all cell lines. In contrast only KMS20 and U266 show strong positivity for Stat 3 phosphorylated. Bcl-xL and Mcl-1 expression is present in all cell lines with no apparent correlation between these proteins and the presence of phosphorylated Stat 3. Bcl-2 is negative in three of the cell lines (KMS20, KMS5, and KMM1). Note that the two cell lines with expression of cyclin D1 show high levels of Bcl-2.
    Cyclin D1, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Upstate Biotechnology Inc cyclin d1
    Effect of microinjection of antisense p27-encoding plasmid into quiescent cells on S-phase entry. (A) Vector plasmids, pcDNA3 and pCMV, or plasmids encoding either antisense p27 (pCMV5-asp27) or CDK4 (pCMV-CDK4 together with a plasmid encoding GFP [pcDNA3-GFP]) were microinjected into serum-starved NIH 3T3 cells (Parental) or their <t>cyclin</t> D1 derivative. BrdU was added to the cultures, and cells were subsequently stained for incorporated BrdU as described in Materials and Methods. The percentages of nuclei staining positive for both BrdU and GFP are shown (filled bars). Also shown is the percent BrdU incorporation for uninjected cells (open bars). Shown are the means plus standard errors for three independent experiments. (B) Same as panel A except that two clonal derivatives of the NIH 3T3-cyclin D1 lines, A2 and C3, were used. The results are for one experiment.
    Cyclin D1, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 93/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Epitomics cyclin d1
    In the experimental arm, overall survival (3A), disease-free survival (3B), locoregional recurrence-free survival (3C), and distant metastasis-free survival (3D) in OSCC patients with low and high <t>cyclin</t> D1 expression.
    Cyclin D1, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cyclin d1
    Effects of a dominant negative shuttling-deficient hnRNP A1 mutant on Akt-dependent <t>cyclin</t> D1 and c- myc IRES activity following rapamycin exposure. A , expression of the NLS-A1-HA mutant in U87 cells. Immunofluorescence microscopy of untransduced (mock-infected,
    Cyclin D1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 565 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen cyclin d1
    Nuclear localization of <t>cyclin</t> D1 is impaired in p27 −/− PMECs. (A) Western analysis of cytoplasmic and nuclear extracts from infected PMECs with antibodies against cyclin D1, p27, and PCNA. (B) Immunofluorescence analysis was used to detect cellular localization of cyclin D1 in infected PMECs. DAPI staining of nuclei is pictured directly below the corresponding cyclin D1 immunofluorescence. Magnification, ×400. Values shown represent the percentage of total nuclei that were positive for cyclin D1 staining. A total of 500 nuclei were counted per experimental condition.
    Cyclin D1, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies cyclin d1
    Proteome analysis of <t>cyclin</t> D1 conditioned medium identifies inflammatory cytokines and dendritic cell maturation pathways A. Venn diagram comparing the overlap of proteins in secretomes of hTERT-control stroma and hTERT-cyclin D1 stroma cells ( n = 3 for each condition). B. Hierarchical clustering heat map of hTERT-control stroma and hTERT-cyclin D1 stroma secretomes based on LFQ intensities of identified proteins. C. Volcano plot of proteins identified in secretome analysis. Log2 ratios of LFQ intensities in hTERT-cyclin D1 stroma vs . hTERT-control stroma were plotted against negative log10 Student’s t -test p-values. Significantly changed proteins, defined as absolute fold-change > 2 and p
    Cyclin D1, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti cyclind1
    Proteome analysis of <t>cyclin</t> D1 conditioned medium identifies inflammatory cytokines and dendritic cell maturation pathways A. Venn diagram comparing the overlap of proteins in secretomes of hTERT-control stroma and hTERT-cyclin D1 stroma cells ( n = 3 for each condition). B. Hierarchical clustering heat map of hTERT-control stroma and hTERT-cyclin D1 stroma secretomes based on LFQ intensities of identified proteins. C. Volcano plot of proteins identified in secretome analysis. Log2 ratios of LFQ intensities in hTERT-cyclin D1 stroma vs . hTERT-control stroma were plotted against negative log10 Student’s t -test p-values. Significantly changed proteins, defined as absolute fold-change > 2 and p
    Anti Cyclind1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 444 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti cyclin d1 antibody sp4
    Proteome analysis of <t>cyclin</t> D1 conditioned medium identifies inflammatory cytokines and dendritic cell maturation pathways A. Venn diagram comparing the overlap of proteins in secretomes of hTERT-control stroma and hTERT-cyclin D1 stroma cells ( n = 3 for each condition). B. Hierarchical clustering heat map of hTERT-control stroma and hTERT-cyclin D1 stroma secretomes based on LFQ intensities of identified proteins. C. Volcano plot of proteins identified in secretome analysis. Log2 ratios of LFQ intensities in hTERT-cyclin D1 stroma vs . hTERT-control stroma were plotted against negative log10 Student’s t -test p-values. Significantly changed proteins, defined as absolute fold-change > 2 and p
    Anti Cyclin D1 Antibody Sp4, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore anti cyclin d1
    Proposed Mechanism of PGV-1 Enhanced S-phase Block and DNA Damage by 5-FU. In a sensitive cancer cell, 5-FU is metabolized into active metabolite 5’-dUMP. It interfere the DNA synthesis and causes apoptosis induced by DNA damage (A). In WiDr cell, 5-FU treatment stimulated NF-κB activation then increased <t>cyclin</t> D1 level. High level of cyclin D1 at S-phase leads to repression of DNA synthesis thus it blocks 5-FU effect (B). In the presence of PGV-1, an inhibitor of NF-κB, cyclin D1 level significantly suppressed and leads to the enhancement of S-phase block by 5-FU (C).
    Anti Cyclin D1, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 260 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher anti cyclin d1
    PTL decreased the viability of C918 cells by arresting G1 phase A: PI staining kit was used to measure the percentage of cell number at cell cycle S, G1, and G2 phase in C918 cells treat with different concentration of PTL. B, C: The relative mRNAs and protein expressions of P21 and <t>Cyclin</t> D1 were detected by qRT-PCR (B) and Western blot (C) assays, respectively. GAPDH served as an internal control. Quality one was applied to measure and count the gray value. a P
    Anti Cyclin D1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Down-regulation of STAT3 regulates the expression of apoptotic and cell cycle related proteins in ECA109 cells. a Cyclin D1, c-Myc, Caspase-3, cleaved Caspase-3 proteins were analyzed by Western blotting in ECA109 cells after transfection with pSi-Scramble and pSi-STAT3. GAPDH was used as loading control. b Quantitative levels of aforementioned proteins against GAPDH. All data are represented as the mean ± SE, *P

    Journal: Cancer Cell International

    Article Title: Down-regulation of STAT3 induces the apoptosis and G1 cell cycle arrest in esophageal carcinoma ECA109 cells

    doi: 10.1186/s12935-018-0549-4

    Figure Lengend Snippet: Down-regulation of STAT3 regulates the expression of apoptotic and cell cycle related proteins in ECA109 cells. a Cyclin D1, c-Myc, Caspase-3, cleaved Caspase-3 proteins were analyzed by Western blotting in ECA109 cells after transfection with pSi-Scramble and pSi-STAT3. GAPDH was used as loading control. b Quantitative levels of aforementioned proteins against GAPDH. All data are represented as the mean ± SE, *P

    Article Snippet: Primary antibodies of Cyclin D1, cleaved Caspase-3 (17 kDa) and enhanced chemiluminescence reagent were purchased from Pierce Biotechnology (Rockford, IL, USA).

    Techniques: Expressing, Western Blot, Transfection

    Expression of apoptosis-associated genes and cell cycle-associated genes in NB4 cells treated with different concentrations of sorafenib for 48 h. (A) Semi-quantitative polymerase chain reaction and (B) western blot analysis demonstrated that sorafenib increased the expression of caspase-8 and caspase-3, and decreased the expression of MCL1 and cyclin D1. *P

    Journal: Oncology Letters

    Article Title: Sorafenib inhibited cell growth through the MEK/ERK signaling pathway in acute promyelocytic leukemia cells

    doi: 10.3892/ol.2018.8010

    Figure Lengend Snippet: Expression of apoptosis-associated genes and cell cycle-associated genes in NB4 cells treated with different concentrations of sorafenib for 48 h. (A) Semi-quantitative polymerase chain reaction and (B) western blot analysis demonstrated that sorafenib increased the expression of caspase-8 and caspase-3, and decreased the expression of MCL1 and cyclin D1. *P

    Article Snippet: The membranes were subsequently blocked with 5% skim milk for 2 h at room temperature, and incubated with rabbit anti-rat MCL1 (cat. no. SAB4501843), caspase-3 (cat. no. C8487), caspase-8 (cat. no. C2976), cyclin D1 (cat. no. SAB4503501), MEK (cat. no. SAB4501863), phosphorylated (P)-MEK (cat. no. M7683), ERK (cat. no. M7556) or P-ERK (cat. no. M7933) polyclonal antibodies (1:500; Sigma-Aldrich; Merck KGaA), or rabbit anti-rat β-actin monoclonal antibody (cat. no. SAB5500001; 1:1,000, Sigma-Aldrich; Merck KGaA) at 4°C overnight. β-actin was used as an internal control.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Cyclin D1 does not accumulate in Fbxo4 −/− MEFs. (A) Growth curves for Fbxo4 +/+ and Fbxo4 −/− MEFs. Data are means ± SD of values obtained from three MEF preparations of each genotype. (B) Immunoblot analysis of cyclin

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Cyclin D1 does not accumulate in Fbxo4 −/− MEFs. (A) Growth curves for Fbxo4 +/+ and Fbxo4 −/− MEFs. Data are means ± SD of values obtained from three MEF preparations of each genotype. (B) Immunoblot analysis of cyclin

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques:

    Depletion of Fbxo4 does not affect cyclin D1 function. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for Fbxo4 +/+ and Fbxo4 −/− MEFs incubated with cycloheximide in S phase. Cells were released from G 0 phase

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Depletion of Fbxo4 does not affect cyclin D1 function. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for Fbxo4 +/+ and Fbxo4 −/− MEFs incubated with cycloheximide in S phase. Cells were released from G 0 phase

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Incubation

    Cyclin D1 is degraded in the absence of Fbxo4, Fbxw8, Skp2, and Fbxo31. (A) Quantitative RT-PCR analysis of Fbxo31 mRNA in Fbxo4 +/+ ; Fbxw8 +/+ and Fbxo4 −/− ; Fbxw8 −/− MEFs infected with a retroviral vector encoding EGFP or

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Cyclin D1 is degraded in the absence of Fbxo4, Fbxw8, Skp2, and Fbxo31. (A) Quantitative RT-PCR analysis of Fbxo31 mRNA in Fbxo4 +/+ ; Fbxw8 +/+ and Fbxo4 −/− ; Fbxw8 −/− MEFs infected with a retroviral vector encoding EGFP or

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation

    Cyclin D1 does not accumulate in Fbxo4 −/− ; Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for Fbxo4 +/+ ; Fbxw8 +/+ and Fbxo4 −/− ; Fbxw8 −/− MEFs incubated

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Cyclin D1 does not accumulate in Fbxo4 −/− ; Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for Fbxo4 +/+ ; Fbxw8 +/+ and Fbxo4 −/− ; Fbxw8 −/− MEFs incubated

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Incubation

    The absence of Fbxo4, Fbxo31, or APC2 does not affect the stability of cyclin D1 after DNA damage. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for NIH 3T3 cells infected with a retroviral vector encoding control (EGFP) or

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: The absence of Fbxo4, Fbxo31, or APC2 does not affect the stability of cyclin D1 after DNA damage. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for NIH 3T3 cells infected with a retroviral vector encoding control (EGFP) or

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Infection, Plasmid Preparation

    Cyclin D1 does not accumulate in Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for MEFs from Fbxw8 +/+ and Fbxw8 −/− mice incubated with cycloheximide (25 μg/ml) for the

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Cyclin D1 does not accumulate in Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for MEFs from Fbxw8 +/+ and Fbxw8 −/− mice incubated with cycloheximide (25 μg/ml) for the

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Mouse Assay, Incubation

    Interactions between ubiquitin ligases and cyclin D1. (A) Lysates of NIH 3T3 cells infected with a retroviral vector encoding FLAG-tagged Fbxo4, Fbxw8, Skp2, Fbxo31, β-TrCP1, Cdh1, or CDK4 or subjected to mock infection were subjected to immunoprecipitation

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Interactions between ubiquitin ligases and cyclin D1. (A) Lysates of NIH 3T3 cells infected with a retroviral vector encoding FLAG-tagged Fbxo4, Fbxw8, Skp2, Fbxo31, β-TrCP1, Cdh1, or CDK4 or subjected to mock infection were subjected to immunoprecipitation

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Infection, Plasmid Preparation, Immunoprecipitation

    Generation and phenotype of Fbxo4 −/− mice. (A) Immunoblot (IB) analysis of cyclin D1 and HSP90 (loading control) in NIH 3T3 cells incubated with cycloheximide (CHX; 25 μg/ml) for the indicated times in the absence or presence of

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Generation and phenotype of Fbxo4 −/− mice. (A) Immunoblot (IB) analysis of cyclin D1 and HSP90 (loading control) in NIH 3T3 cells incubated with cycloheximide (CHX; 25 μg/ml) for the indicated times in the absence or presence of

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Mouse Assay, Incubation

    Cyclin D1 does not accumulate in Skp2-depleted Fbxo4 −/− ; Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for MEFs from Skp2 +/+ and Skp2 −/− mice incubated with cycloheximide

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Cyclin D1 does not accumulate in Skp2-depleted Fbxo4 −/− ; Fbxw8 −/− MEFs. (A) Immunoblot analysis of cyclin D1 (left) and its quantification (right) for MEFs from Skp2 +/+ and Skp2 −/− mice incubated with cycloheximide

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Mouse Assay, Incubation

    Acute depletion of Fbxo4 or Fbxw8 does not affect the half-life of cyclin D1. (A) Quantitative RT-PCR analysis of Fbxo4 mRNA in NIH 3T3 cells infected with a retroviral vector encoding control (EGFP) or Fbxo4 shRNAs. Data are means ± SD of triplicates

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Acute depletion of Fbxo4 or Fbxw8 does not affect the half-life of cyclin D1. (A) Quantitative RT-PCR analysis of Fbxo4 mRNA in NIH 3T3 cells infected with a retroviral vector encoding control (EGFP) or Fbxo4 shRNAs. Data are means ± SD of triplicates

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation

    Inhibition of Cul1 function does not affect the stability of cyclin D1. (A) Immunoblot analysis with antibodies to the hemagglutinin epitope (HA; top) or to p27 and cyclin E1 (bottom left), as well as quantification of p27 (bottom right), for NIH 3T3

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Reevaluation of the Role of F-Box Proteins in Cyclin D1 Degradation

    doi: 10.1128/MCB.06570-11

    Figure Lengend Snippet: Inhibition of Cul1 function does not affect the stability of cyclin D1. (A) Immunoblot analysis with antibodies to the hemagglutinin epitope (HA; top) or to p27 and cyclin E1 (bottom left), as well as quantification of p27 (bottom right), for NIH 3T3

    Article Snippet: The half-life of cyclin D1 determined by pulse-chase analysis was almost identical to that determined by cycloheximide chase analysis, and the stability of cyclin D1 also did not differ between Fbxo4 +/+ and Fbxo4 −/− MEFs with this approach , suggesting that the half-life of cyclin D1 determined with the cycloheximide chase assay represents the physiological turnover rate of the protein.

    Techniques: Inhibition

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    The expression of AKAP95, CyclinE1, CyclinD1 and Cx43, and their association with clinical and pathological parameters

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The expression of AKAP95, CyclinE1, CyclinD1 and Cx43, and their association with clinical and pathological parameters

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques: Expressing

    The expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal carcinoma and pericarcinoma tissues

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal carcinoma and pericarcinoma tissues

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques: Expressing

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    Expression of CyclinD1 in pericarcinoma and esophageal carcinoma tissues. A. Pericarcinoma tissues express CyclinD1 negatively. B. CyclinD1 is expressed in the cytoplasm of pericarcinoma tissues. C. CyclinD1 is expressed in the cytoplasm and poorly expressed

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: Expression of CyclinD1 in pericarcinoma and esophageal carcinoma tissues. A. Pericarcinoma tissues express CyclinD1 negatively. B. CyclinD1 is expressed in the cytoplasm of pericarcinoma tissues. C. CyclinD1 is expressed in the cytoplasm and poorly expressed

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques: Expressing

    The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Journal: International Journal of Clinical and Experimental Medicine

    Article Title: Expression of AKAP95, Cx43, CyclinE1 and CyclinD1 in esophageal cancer and their association with the clinical and pathological parameters

    doi:

    Figure Lengend Snippet: The correlation among AKAP95, CyclinE1, CyclinD1 and Cx43 in esophageal carcinoma

    Article Snippet: The antibodies included mouse anti-human CyclinE1 (sc-247, 1:300), AKAP95 (sc-100643, 1:150), Cx43 (sc-13558, 1:300) and CyclinD1 (sc-20044, 1:300) monoclonal antibodies (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA).

    Techniques:

    Effects of downregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, cyclin A2, p21 and cyclin D1 protein expression in each group of SGC-7901 cells. (B) Comparison of protein levels in each group of SGC-7901 cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of SGC-7901 cells. (D) Comparison of protein levels in each group of SGC-7901 cells. **P

    Journal: Oncology Reports

    Article Title: MicroRNA-181a promotes cell proliferation and inhibits apoptosis in gastric cancer by targeting RASSF1A

    doi: 10.3892/or.2018.6632

    Figure Lengend Snippet: Effects of downregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, cyclin A2, p21 and cyclin D1 protein expression in each group of SGC-7901 cells. (B) Comparison of protein levels in each group of SGC-7901 cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of SGC-7901 cells. (D) Comparison of protein levels in each group of SGC-7901 cells. **P

    Article Snippet: Anti-cyclin A2 (mouse IgG; cat. no. 4656) and cyclin D1 (rabbit IgG; cat. no. 2922) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    Effects of upregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, cyclin A2, p21 and cyclin D1 protein expression in each group of AGS cells. (B) Comparison of protein levels in each group of AGS cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of AGS cells. (D) Comparison of protein levels in each group of AGS cells. **P

    Journal: Oncology Reports

    Article Title: MicroRNA-181a promotes cell proliferation and inhibits apoptosis in gastric cancer by targeting RASSF1A

    doi: 10.3892/or.2018.6632

    Figure Lengend Snippet: Effects of upregulated miR-181a on the expression of tumorigenesis-associated proteins. (A) Western blot band of CDC25A, cyclin A2, p21 and cyclin D1 protein expression in each group of AGS cells. (B) Comparison of protein levels in each group of AGS cells. (C) Western blot band of Bcl-2 and Bax protein expression in each group of AGS cells. (D) Comparison of protein levels in each group of AGS cells. **P

    Article Snippet: Anti-cyclin A2 (mouse IgG; cat. no. 4656) and cyclin D1 (rabbit IgG; cat. no. 2922) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

    Techniques: Expressing, Western Blot

    USP14 inhibition or silencing blocks G 0 /G 1 to S phase transition in breast cancer cells. a Fluorescence-activated cell sorting analysis (FACS) was performed on the indicated breast cancer cells exposed to IU1 for 48 h. ( n = 3). b The percentage of cells in each population was calculated. Mean ± S.D. ( n = 3). c FACS analysis was performed on the indicated breast cancer cells that stably expressed USP14 shRNA or control shRNA for 48 h. The percentage of cells in each population was calculated. Mean ± S.D. ( n = 3). d Protein lysates were collected from the indicated breast cancer cells treated with IU1 for 48 h. Western blot assay was used to detect CDK4, CDK2, and cyclin D1 protein expression. GAPDH was used as an internal control. e Protein lysates were collected from the indicated breast cancer cells stably expressing USP14 shRNA or control shRNA. Western blot assay was used to detect CDK4, CDK2, and cyclin D1 expression. GAPDH was used as an internal control

    Journal: Oncogene

    Article Title: Growth arrest and apoptosis induction in androgen receptor-positive human breast cancer cells by inhibition of USP14-mediated androgen receptor deubiquitination

    doi: 10.1038/s41388-017-0069-z

    Figure Lengend Snippet: USP14 inhibition or silencing blocks G 0 /G 1 to S phase transition in breast cancer cells. a Fluorescence-activated cell sorting analysis (FACS) was performed on the indicated breast cancer cells exposed to IU1 for 48 h. ( n = 3). b The percentage of cells in each population was calculated. Mean ± S.D. ( n = 3). c FACS analysis was performed on the indicated breast cancer cells that stably expressed USP14 shRNA or control shRNA for 48 h. The percentage of cells in each population was calculated. Mean ± S.D. ( n = 3). d Protein lysates were collected from the indicated breast cancer cells treated with IU1 for 48 h. Western blot assay was used to detect CDK4, CDK2, and cyclin D1 protein expression. GAPDH was used as an internal control. e Protein lysates were collected from the indicated breast cancer cells stably expressing USP14 shRNA or control shRNA. Western blot assay was used to detect CDK4, CDK2, and cyclin D1 expression. GAPDH was used as an internal control

    Article Snippet: Antibodies were from the following corporations: anti-GAPDH (MB001), anti-CDK2 (BS1050), anti-GFP (BSAP0675M), anti-Bcl-2 (BS70205) (Bioworld Technology, Inc., Louis Park, MN, USA); anti-Ubiquitin (catalog no. 3936), anti-PARP (9532), anti-CDK4 (12790), anti-USP14 (11931), anti-Lamin B (13435), anti-HSP90 (4877), HA-tag (3724), anti-K48-ub (12805), anti-cyclin D1 (2922) (Cell Signaling Technology, Beverly, MA, USA); anti-AR (ab108341), anti-UCHL5 (ab124931) (Abcam, USA).

    Techniques: Inhibition, Sublimation, Fluorescence, FACS, Stable Transfection, shRNA, Western Blot, Expressing

    Inhibition of PI3K/Akt totally blocked the promotion of HC11 proliferation induced by OA ( A ) Effect of Wortmannin (WT), an inhibitor of PI3K, on the proliferation of HC11 after a 4-day incubation was determined by MTT assay. ( B ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or 100 nM WT. ( C ) Representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or 100 nM WT. Scale bar = 100 μm. ( D ) Analysis of the relative fluorescence intensity in panel ( C). (E ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1 and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 100 nM WT. β-actin was used as the loading control. ( F ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1, and p21, and the intensities of the bands were expressed as the arbitrary units. ** P

    Journal: Oncotarget

    Article Title: Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway

    doi: 10.18632/oncotarget.24204

    Figure Lengend Snippet: Inhibition of PI3K/Akt totally blocked the promotion of HC11 proliferation induced by OA ( A ) Effect of Wortmannin (WT), an inhibitor of PI3K, on the proliferation of HC11 after a 4-day incubation was determined by MTT assay. ( B ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or 100 nM WT. ( C ) Representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or 100 nM WT. Scale bar = 100 μm. ( D ) Analysis of the relative fluorescence intensity in panel ( C). (E ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1 and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 100 nM WT. β-actin was used as the loading control. ( F ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1, and p21, and the intensities of the bands were expressed as the arbitrary units. ** P

    Article Snippet: Antibodies against Cyclin D1 (#2922), p21 (#2947), phospho-Akt Ser473 (p-Akt Ser473 , #4060) and Akt (#9272) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA).

    Techniques: Inhibition, Incubation, MTT Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

    Knockdown of CD36 eliminated the enhancement of HC11 proliferation induced by OA ( A , B ) Effects of 100 μM OA and/or CD36 siRNA on HC11 proliferation was determined using MTT analysis ( A ) and EdU incorporation assay ( B ). The nuclei were stained with Hoechst and the scale bar = 200 μm. ( C ) Analysis of EdU positive cell percentage in panel B. ( D ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or CD36 siRNA. ( E ) The representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or CD36 siRNA. Scale bar = 100 μm. ( F ) Western blot analysis of CD36, Cyclin D1 and p21 in HC11 after a 4-day culture. β-actin was used as the loading control. ( G ) Mean ± SEM of immunoblotting bands of CD36, Cyclin D1, and p21. The intensities of the bands were expressed as the arbitrary units. ** P

    Journal: Oncotarget

    Article Title: Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway

    doi: 10.18632/oncotarget.24204

    Figure Lengend Snippet: Knockdown of CD36 eliminated the enhancement of HC11 proliferation induced by OA ( A , B ) Effects of 100 μM OA and/or CD36 siRNA on HC11 proliferation was determined using MTT analysis ( A ) and EdU incorporation assay ( B ). The nuclei were stained with Hoechst and the scale bar = 200 μm. ( C ) Analysis of EdU positive cell percentage in panel B. ( D ) The relative mRNA expression level of Cyclin D1, Cyclin D3, and PCNA in response to 100 μM OA and/or CD36 siRNA. ( E ) The representative immunofluorescence staining of Cyclin D1 and p21 in the presence of 100 μM OA and/or CD36 siRNA. Scale bar = 100 μm. ( F ) Western blot analysis of CD36, Cyclin D1 and p21 in HC11 after a 4-day culture. β-actin was used as the loading control. ( G ) Mean ± SEM of immunoblotting bands of CD36, Cyclin D1, and p21. The intensities of the bands were expressed as the arbitrary units. ** P

    Article Snippet: Antibodies against Cyclin D1 (#2922), p21 (#2947), phospho-Akt Ser473 (p-Akt Ser473 , #4060) and Akt (#9272) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA).

    Techniques: MTT Assay, Staining, Expressing, Immunofluorescence, Western Blot

    Chelation of [Ca 2+ ] i reversed the OA-induced activation of PI3K/Akt and promotion of HC11 proliferation ( A ) Effects of 100 μM OA and/or 2 μM BAPTA-AM on HC11 proliferation by using MTT analysis. ( B ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1, and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 2 μM BAPTA-AM. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1 and p21. The intensities of the bands were expressed as the arbitrary units. ** P

    Journal: Oncotarget

    Article Title: Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway

    doi: 10.18632/oncotarget.24204

    Figure Lengend Snippet: Chelation of [Ca 2+ ] i reversed the OA-induced activation of PI3K/Akt and promotion of HC11 proliferation ( A ) Effects of 100 μM OA and/or 2 μM BAPTA-AM on HC11 proliferation by using MTT analysis. ( B ) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, Cyclin D1, and p21 in HC11 after a 4-day culture in the presence of 100 μM OA and/or 2 μM BAPTA-AM. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of p-PI3K/PI3K, p-Akt/Akt, Cyclin D1 and p21. The intensities of the bands were expressed as the arbitrary units. ** P

    Article Snippet: Antibodies against Cyclin D1 (#2922), p21 (#2947), phospho-Akt Ser473 (p-Akt Ser473 , #4060) and Akt (#9272) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA).

    Techniques: Activation Assay, MTT Assay, Western Blot

    OA enhanced the proliferation of HC11 and the expression of CD36 ( A ) Effect of various concentrations of OA (0, 12.5, 25, 50, 100, and 200 μM) on the proliferation of HC11 after a 4-day culture was determined by MTT analysis. ( B ) Western blot analysis of proliferative markers (Cyclin D1 and p21) and CD36 in HC11 after a 4-day culture. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of CD36, Cyclin D1 and p21, and the intensities of the bands were expressed as the arbitrary units. * P

    Journal: Oncotarget

    Article Title: Oleic acid stimulates HC11 mammary epithelial cells proliferation and mammary gland development in peripubertal mice through activation of CD36-Ca2+ and PI3K/Akt signaling pathway

    doi: 10.18632/oncotarget.24204

    Figure Lengend Snippet: OA enhanced the proliferation of HC11 and the expression of CD36 ( A ) Effect of various concentrations of OA (0, 12.5, 25, 50, 100, and 200 μM) on the proliferation of HC11 after a 4-day culture was determined by MTT analysis. ( B ) Western blot analysis of proliferative markers (Cyclin D1 and p21) and CD36 in HC11 after a 4-day culture. β-actin was used as the loading control. ( C ) Mean ± SEM of immunoblotting bands of CD36, Cyclin D1 and p21, and the intensities of the bands were expressed as the arbitrary units. * P

    Article Snippet: Antibodies against Cyclin D1 (#2922), p21 (#2947), phospho-Akt Ser473 (p-Akt Ser473 , #4060) and Akt (#9272) were purchased from Cell Signaling Technology Inc (Danvers, MA, USA).

    Techniques: Expressing, MTT Assay, Western Blot

    Cyclin D1-CDK4 regulates gluconeogenesis in primary hepatocytes and in whole animals. a) Western-blot analysis of endogenous, forskolin-induced (Fsk) or adenovirally overexpressed (O/E) PGC-1α. Nuclear extracts of primary hepatocytes were used to immunoprecipitate PGC-1α. Cells were infected with GFP or PGC-1α 48hr prior to harvest. 10μM forskolin was added for 2hr before harvest. b) PD 0332991 increases forskolin-induced gluconeogenic gene expression. Primary hepatocytes were treated with 10μM forskolin for 1.5hr following 3hr of starvation medium incubation while 1μM PD 0332991 was added overnight (one-way ANOVA with Tukey post test, n=3). c) CDK4 knockdown increases forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). d) Cyclin D1 wild-type, but not cyclin D1 K112E mutant, suppresses forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). e) Phosphorylation of GCN5, FoxO1 N-terminus, FoxO1 C-terminus, FoxO3A and PGC-1α SR domain by cyclin D1-CDK4. f) PGC-1α knockdown blocks the increase of forskolin-induced gluconeogenic genes by fascaplysin in HepG2 cells. PGC-1α knockdown or a negative control HepG2 cells were treated with 30μM forskolin and 1μM fascaplysin overnight (one-way ANOVA with Tukey post test, n=3). g) GCN5 knockdown blunts the increase of gluconeogenic gene expression caused by CDK4 knockdown. qRT-PCR analysis of Pck1 and Gcn5 and western-blot of CDK4 and GCN5 knockdown are shown. All cells were infected with PGC-1α adenoviruses (one-way ANOVA with Tukey post test, n=15). h) PD 0332991 increases gluconeogenic genes when combined with GCN5 wild-type (WT) overexpression, but not with GCN5 T272A/S372A (AA) mutant. GFP infected cells shown as a comparison to GCN5 overexpressing cells. All cells were infected with PGC-1α adenoviruses (two-tailed unpaired t-test, n=6). I-j) Insulin levels measured from serum and western-blot analysis of Rb and AKT using nuclear (N) and cytoplasmic (C) liver extracts from mice treated with vehicle or 150mg/kg PD 0332991, shown in Fig. 2lm ( i : two-tailed unpaired t-test, n=18/GFP, n=17/PD 0332991). k) Levels of cyclin D1 and Rb phosphorylation in GFP or Cyclin D1 T286A tail-vein injected mice, shown in Fig. 2n-o . Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 regulates gluconeogenesis in primary hepatocytes and in whole animals. a) Western-blot analysis of endogenous, forskolin-induced (Fsk) or adenovirally overexpressed (O/E) PGC-1α. Nuclear extracts of primary hepatocytes were used to immunoprecipitate PGC-1α. Cells were infected with GFP or PGC-1α 48hr prior to harvest. 10μM forskolin was added for 2hr before harvest. b) PD 0332991 increases forskolin-induced gluconeogenic gene expression. Primary hepatocytes were treated with 10μM forskolin for 1.5hr following 3hr of starvation medium incubation while 1μM PD 0332991 was added overnight (one-way ANOVA with Tukey post test, n=3). c) CDK4 knockdown increases forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). d) Cyclin D1 wild-type, but not cyclin D1 K112E mutant, suppresses forskolin-induced gluconeogenic gene expression (one-way ANOVA with Tukey post test, n=3). e) Phosphorylation of GCN5, FoxO1 N-terminus, FoxO1 C-terminus, FoxO3A and PGC-1α SR domain by cyclin D1-CDK4. f) PGC-1α knockdown blocks the increase of forskolin-induced gluconeogenic genes by fascaplysin in HepG2 cells. PGC-1α knockdown or a negative control HepG2 cells were treated with 30μM forskolin and 1μM fascaplysin overnight (one-way ANOVA with Tukey post test, n=3). g) GCN5 knockdown blunts the increase of gluconeogenic gene expression caused by CDK4 knockdown. qRT-PCR analysis of Pck1 and Gcn5 and western-blot of CDK4 and GCN5 knockdown are shown. All cells were infected with PGC-1α adenoviruses (one-way ANOVA with Tukey post test, n=15). h) PD 0332991 increases gluconeogenic genes when combined with GCN5 wild-type (WT) overexpression, but not with GCN5 T272A/S372A (AA) mutant. GFP infected cells shown as a comparison to GCN5 overexpressing cells. All cells were infected with PGC-1α adenoviruses (two-tailed unpaired t-test, n=6). I-j) Insulin levels measured from serum and western-blot analysis of Rb and AKT using nuclear (N) and cytoplasmic (C) liver extracts from mice treated with vehicle or 150mg/kg PD 0332991, shown in Fig. 2lm ( i : two-tailed unpaired t-test, n=18/GFP, n=17/PD 0332991). k) Levels of cyclin D1 and Rb phosphorylation in GFP or Cyclin D1 T286A tail-vein injected mice, shown in Fig. 2n-o . Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Western Blot, Pyrolysis Gas Chromatography, Infection, Expressing, Incubation, Mutagenesis, Negative Control, Quantitative RT-PCR, Over Expression, Two Tailed Test, Mouse Assay, Injection

    Cyclin D1-CDK4 is regulated by insulin/GSK3β and hepatic cyclin D1 deletion causes increased gluconeogenesis and glycemia upon refeeding. a) Cyclin D1 gene expression is increased during refeeding (one-way ANOVA with Tukey post test, n=3/fast and10hr refed, n=4/4hr refed). b) Cyclin D1 protein and Rb phosphorylation are induced upon refeeding (N=nuclear and C=cytoplasmic liver extracts). c) Phosphorylation of GCN5 is increased upon refeeding. GCN5 was immunoprecipitated using anti-phosphoS*P (pS*P) antibody from livers infected with GFP or GCN5 adenovirus. d) Hepatocytes ploidy does not change upon fasting and refeeding (n=5). e) Ki-67 staining in liver shows no differences upon fasting and refeeding. Small intestine was used as a positive control. f) Nuclear cyclin D1 protein level is increased upon insulin and GSK3β inhibitors treatment in primary hepatocytes. All cells were infected with cyclin D1 wild-type adenovirus. g) Insulin and GSK3β inhibitors suppress gluconeogenic gene expression. All cells were infected with PGC-1α adenovirus (oneway ANOVA with Tukey post test, n=6). h) Phosphorylation of GCN5 is induced by insulin and blunted by PD 0332991 treatment. GCN5 was overexpressed whereas GFP was used as a negative control. i) Cyclin D1 is transcriptionally induced by dietary intake of amino acids. Mice were fasted overnight or refed 4hr with chow diet, empty calorie, glucose or glucose and amino acids diet (one-way ANOVA with Tukey post test, n=5). j-k) Liver-specific cyclin D1 KO (D1 LKO) mice exhibit no differences on gluconeogenic gene expression and glycemia during fasting. l-m) D1 LKO mice display increased gluconeogenic gene expression and glycemia upon 4hr refeeding (one-way ANOVA with Tukey post test, combined 4 cohorts of n=3/fasting, n=5/refeeding). n) D1 LKO mice show mild glucose intolerance (two-way ANOVA, multiple comparison, n=11/WT1, n=7/WT2, n=10/D1 LKO). o) D1 LKO mice exhibit mild insulin intolerance (two-way ANOVA, significant interaction, multiple comparison, n=10/WT1, n=7/WT2, n=9/D1 LKO). Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 is regulated by insulin/GSK3β and hepatic cyclin D1 deletion causes increased gluconeogenesis and glycemia upon refeeding. a) Cyclin D1 gene expression is increased during refeeding (one-way ANOVA with Tukey post test, n=3/fast and10hr refed, n=4/4hr refed). b) Cyclin D1 protein and Rb phosphorylation are induced upon refeeding (N=nuclear and C=cytoplasmic liver extracts). c) Phosphorylation of GCN5 is increased upon refeeding. GCN5 was immunoprecipitated using anti-phosphoS*P (pS*P) antibody from livers infected with GFP or GCN5 adenovirus. d) Hepatocytes ploidy does not change upon fasting and refeeding (n=5). e) Ki-67 staining in liver shows no differences upon fasting and refeeding. Small intestine was used as a positive control. f) Nuclear cyclin D1 protein level is increased upon insulin and GSK3β inhibitors treatment in primary hepatocytes. All cells were infected with cyclin D1 wild-type adenovirus. g) Insulin and GSK3β inhibitors suppress gluconeogenic gene expression. All cells were infected with PGC-1α adenovirus (oneway ANOVA with Tukey post test, n=6). h) Phosphorylation of GCN5 is induced by insulin and blunted by PD 0332991 treatment. GCN5 was overexpressed whereas GFP was used as a negative control. i) Cyclin D1 is transcriptionally induced by dietary intake of amino acids. Mice were fasted overnight or refed 4hr with chow diet, empty calorie, glucose or glucose and amino acids diet (one-way ANOVA with Tukey post test, n=5). j-k) Liver-specific cyclin D1 KO (D1 LKO) mice exhibit no differences on gluconeogenic gene expression and glycemia during fasting. l-m) D1 LKO mice display increased gluconeogenic gene expression and glycemia upon 4hr refeeding (one-way ANOVA with Tukey post test, combined 4 cohorts of n=3/fasting, n=5/refeeding). n) D1 LKO mice show mild glucose intolerance (two-way ANOVA, multiple comparison, n=11/WT1, n=7/WT2, n=10/D1 LKO). o) D1 LKO mice exhibit mild insulin intolerance (two-way ANOVA, significant interaction, multiple comparison, n=10/WT1, n=7/WT2, n=9/D1 LKO). Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Expressing, Immunoprecipitation, Infection, Staining, Positive Control, Pyrolysis Gas Chromatography, Negative Control, Mouse Assay

    Cyclin D1-CDK4 modulates PGC-1α acetylation through GCN5. a) Fascaplysin decreases PGC-1α acetylation in dose-dependent manner. Dose-dependent response of PGC-1α acetylation treated with fascaplysin concentrations ranging from 31.25nM to 8μM. IC 50 value was calculated using three independent measurements from the assay described in Extended Data Fig. 1 a . b) Chemical structures of fascaplysin and PD 0332991. c) CDK4 is knockdown by various CDK4 shRNAs used in Fig. 1d . d) Fascaplysin decreases PGC-1α acetylation upon EX527 or trichostatin A treatments. Cells were treated for 8hr in case of 1μM fascaplysin and 4hr in case of 1μM EX527 or trichostatin A prior to harvest. DMSO (-) was used as a control treatment. e) Fascpalysin has blunted effect on PCAF-mediated PGC-1α acetylation. f) Ectopically expressed CDK4 and GCN5 interact. As a comparison, PGC-1α, Sirt1 and Sirt6 were used while GFP was overexpressed as a negative control. g) Phosphorylation of GCN5 by cyclin D1-CDK4 complex is reduced by fascaplysin. DMSO or 1μM fascaplysin was added to the kinase reaction. h) In vitro phosphorylation of GST-GCN5 recombinant proteins (1-224aa, 1-386aa, 1-553aa, 1-837aa) by cyclin D1-CDK4 and the protein level of those fragments. i) GCN5 wild-type (WT), treated with fascaplysin and GCN5 T272A/S372A (AA) mutant immunoprecipitated by anti-phospho-S*P (pS*P) antibody. j) Acetylation of PGC-1α closely follows the amount of PAF65β bound to GCN5. Nuclear extracts of U-2OS overexpressing various amounts of GCN5 were used for western-blot analysis to detect GCN5 and PAF65β. Empty vector was transfected as a negative control. k) Interaction between GCN5 T272A/S372A (AA) and PAF65β is reduced compared to GCN5 wild-type (WT). U-2OS cells were used for western-blot analysis experiments.

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 modulates PGC-1α acetylation through GCN5. a) Fascaplysin decreases PGC-1α acetylation in dose-dependent manner. Dose-dependent response of PGC-1α acetylation treated with fascaplysin concentrations ranging from 31.25nM to 8μM. IC 50 value was calculated using three independent measurements from the assay described in Extended Data Fig. 1 a . b) Chemical structures of fascaplysin and PD 0332991. c) CDK4 is knockdown by various CDK4 shRNAs used in Fig. 1d . d) Fascaplysin decreases PGC-1α acetylation upon EX527 or trichostatin A treatments. Cells were treated for 8hr in case of 1μM fascaplysin and 4hr in case of 1μM EX527 or trichostatin A prior to harvest. DMSO (-) was used as a control treatment. e) Fascpalysin has blunted effect on PCAF-mediated PGC-1α acetylation. f) Ectopically expressed CDK4 and GCN5 interact. As a comparison, PGC-1α, Sirt1 and Sirt6 were used while GFP was overexpressed as a negative control. g) Phosphorylation of GCN5 by cyclin D1-CDK4 complex is reduced by fascaplysin. DMSO or 1μM fascaplysin was added to the kinase reaction. h) In vitro phosphorylation of GST-GCN5 recombinant proteins (1-224aa, 1-386aa, 1-553aa, 1-837aa) by cyclin D1-CDK4 and the protein level of those fragments. i) GCN5 wild-type (WT), treated with fascaplysin and GCN5 T272A/S372A (AA) mutant immunoprecipitated by anti-phospho-S*P (pS*P) antibody. j) Acetylation of PGC-1α closely follows the amount of PAF65β bound to GCN5. Nuclear extracts of U-2OS overexpressing various amounts of GCN5 were used for western-blot analysis to detect GCN5 and PAF65β. Empty vector was transfected as a negative control. k) Interaction between GCN5 T272A/S372A (AA) and PAF65β is reduced compared to GCN5 wild-type (WT). U-2OS cells were used for western-blot analysis experiments.

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Pyrolysis Gas Chromatography, Negative Control, In Vitro, Recombinant, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Transfection

    Cyclin D1-CDK4 regulates gluconeogenesis in primary hepatocytes and whole animals. a-b ) PD 0332991 increases gluconeogenic gene expression and glucose production ( a : one-way ANOVA with Tukey post test, n=3, b : two-tailed unpaired t-test, n=8). c) PGC-1α acetylation is decreased by PD 0332991 treatment. d-e) CDK4 knockdown increases gluconeogenic gene expression and glucose production ( d : oneway ANOVA with Tukey post test, n=6, e : two-tailed unpaired t-test, n=6). f) PGC-1α acetylation is decreased upon CDK4 deletion. g-h) Cyclin D1 wild-type decreases gluconeogenic gene expression, and cyclin D1 wild-type and cyclin D1 T286A, but not cyclin D1 K112E, repress glucose production ( g : one-way ANOVA with Tukey post test, n=3/GFP, PGC-1α, n=6/Cyclin D1 WT and KE, h : one-way ANOVA with Dunnett post test, n=8). i) Overexpression of cyclin D1 wild-type (WT) and T286A (TA), but not cyclin D1 K112E (KE), induces PGC-1α acetylation. j) GCN5 knockdown blunts the increase of glucose production by CDK4 knockdown (two-tailed unpaired t-test, n=8). k) PD 0332991 increases glucose production with GCN5 wild-type (WT), but not with GCN5 T272A/S372A (AA) (one-way ANOVA with Newman-Keuls post test, n=8). l-m) PD 0332991 administration increases Pck1 gene expression and glycemia in mice (two-tailed unpaired t-test, n=18/vehicle, n=17/PD 0332991). n-o) Gluconeogenic gene expression and glycemia are reduced by cyclin D1 T286A overexpression in liver (two-tailed unpaired t-test, n=10/GFP, n=9/D1 T286A). p ) Cyclin D1 T286A overexpression in liver decreases hepatic glucose production capacity assessed by pyruvate tolerance test (two-tailed unpaired t-test, n=20/GFP, n=24/D1 T286A AUC=area under curve). Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 regulates gluconeogenesis in primary hepatocytes and whole animals. a-b ) PD 0332991 increases gluconeogenic gene expression and glucose production ( a : one-way ANOVA with Tukey post test, n=3, b : two-tailed unpaired t-test, n=8). c) PGC-1α acetylation is decreased by PD 0332991 treatment. d-e) CDK4 knockdown increases gluconeogenic gene expression and glucose production ( d : oneway ANOVA with Tukey post test, n=6, e : two-tailed unpaired t-test, n=6). f) PGC-1α acetylation is decreased upon CDK4 deletion. g-h) Cyclin D1 wild-type decreases gluconeogenic gene expression, and cyclin D1 wild-type and cyclin D1 T286A, but not cyclin D1 K112E, repress glucose production ( g : one-way ANOVA with Tukey post test, n=3/GFP, PGC-1α, n=6/Cyclin D1 WT and KE, h : one-way ANOVA with Dunnett post test, n=8). i) Overexpression of cyclin D1 wild-type (WT) and T286A (TA), but not cyclin D1 K112E (KE), induces PGC-1α acetylation. j) GCN5 knockdown blunts the increase of glucose production by CDK4 knockdown (two-tailed unpaired t-test, n=8). k) PD 0332991 increases glucose production with GCN5 wild-type (WT), but not with GCN5 T272A/S372A (AA) (one-way ANOVA with Newman-Keuls post test, n=8). l-m) PD 0332991 administration increases Pck1 gene expression and glycemia in mice (two-tailed unpaired t-test, n=18/vehicle, n=17/PD 0332991). n-o) Gluconeogenic gene expression and glycemia are reduced by cyclin D1 T286A overexpression in liver (two-tailed unpaired t-test, n=10/GFP, n=9/D1 T286A). p ) Cyclin D1 T286A overexpression in liver decreases hepatic glucose production capacity assessed by pyruvate tolerance test (two-tailed unpaired t-test, n=20/GFP, n=24/D1 T286A AUC=area under curve). Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Expressing, Two Tailed Test, Pyrolysis Gas Chromatography, Over Expression, Mouse Assay

    In diabetic hyperinsulinemic mice, cyclin D1-CDK4 is dysregulated and hyperactivation of cyclin D1-CDK4 attenuates the diabetic phenotype. a) Cyclin D1 is chronically elevated in livers of db/db mice (N=nuclear and C=cytoplasmic liver extracts). b-c) Cyclin D1 T286A, but not cyclin D1 K112E, overexpression in liver represses gluconeogenic genes and glycemia in db/db mice (one-way ANOVA, Tukey post test, n=6/GFP, D1 K112E, n=5/D1 T286A). d-h) In db/db mice, cyclin D1 T286A overexpression in liver suppresses hepatic glucose production tested by hyperinsulinemic-euglycemic clamp. d) Plasma glycemia and glucose infusion rate (twoway ANOVA, significant interaction, n=7). e) Body weights. f) Clamped glucose infusion rate. g) Whole-body glucose uptake. h) Hepatic glucose output (average of last 40min values for f-g , two-tailed unpaired t-test for d-h , n=7). Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: In diabetic hyperinsulinemic mice, cyclin D1-CDK4 is dysregulated and hyperactivation of cyclin D1-CDK4 attenuates the diabetic phenotype. a) Cyclin D1 is chronically elevated in livers of db/db mice (N=nuclear and C=cytoplasmic liver extracts). b-c) Cyclin D1 T286A, but not cyclin D1 K112E, overexpression in liver represses gluconeogenic genes and glycemia in db/db mice (one-way ANOVA, Tukey post test, n=6/GFP, D1 K112E, n=5/D1 T286A). d-h) In db/db mice, cyclin D1 T286A overexpression in liver suppresses hepatic glucose production tested by hyperinsulinemic-euglycemic clamp. d) Plasma glycemia and glucose infusion rate (twoway ANOVA, significant interaction, n=7). e) Body weights. f) Clamped glucose infusion rate. g) Whole-body glucose uptake. h) Hepatic glucose output (average of last 40min values for f-g , two-tailed unpaired t-test for d-h , n=7). Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Mouse Assay, Over Expression, Two Tailed Test

    Cyclin D1-CDK4 is regulated by insulin/GSK3β and hepatic specific cyclin D1 deletion causes increased gluconeogenesis and glycemia upon refeeding. a) Cyclin D1 transcripts are increased upon refeeding. qRT-PCR analysis of Ccnd1 , Ccnd2 and Ccne1 gene expression upon overnight fasting, 4hr and 10hr refeeding in BALB/c mice livers (one-way ANOVA with Tukey post test, n=3). b) Cyclin D1 protein is increased upon refeeding. Western-blot analysis of cyclin D1 protein levels and associated signaling pathway upon fasting and refeeding measured from nuclear (N) and cytoplasmic (C) liver extracts from BALB/c mice. c) Cyclin D1-CDK4 kinase activity is increased upon 4hr refeding. In vitro 32 P incorporation into recombinant Rb by immunoprecipitated cyclin D1-CDK4 kinase from whole-cell extracts of overnight fast and 4hr refed livers. d) Western-blot analysis of cyclin D1 protein level and associated signaling pathway upon fasting and refeeding and qRT-PCR analysis of Ccnd1 and Pgc1α mRNA level in various tissues (L=liver, M=skeletal muscle, B=brown adipose tissue, W=epididymal white adipose tissue, and P=pancreas, two-tailed unpaired t-test, n=12/L, n=4/M, B, W, P). e) qRT-PCR analysis of PGC-1α target genes in liver and epididymal white adipose tissues (eWAT) upon vehicle or PD 0332991 treatment (two-tailed unpaired t-test, n=10). f) Ccnb1 and Pcna gene expressions in liver do not change upon fasting and refeeding (n=3/fast and 10hr refed, n=4/4hr refed). g) BrdU incorporation in liver does not change upon fasting and refeeding. Small intestine was used as a positive control. h) Ki-67 staining in liver does not change upon fasting and refeeding following vehicle or PD 0332991 administration. Small intestine used as a positive control. i) Ki-67 staining in liver does not change upon fasting and refeeding following GFP or cyclin D1 T286A tail-vein injection. Small intestine used as a positive control. j) Hepatic ploidy profiles of livers of GFP or cyclin D1 T286A adenovirus tail-vein injected mice do not show significant difference. Ploidy analysis of primary hepatocytes isolated from livers measured by propidium iodide staining and flow cytometry (n=6/fast and 4hr refed, n=4/10hr refed). k) Western-blot analysis of endogenous nuclear (N) and cytoplasmic (C) cyclin D1 protein level upon insulin or GSK3β inhibitors treatments in primary hepatocytes. l) PGC-1α acetylation is increased upon insulin or GSK3β inhibitors treatment in primary hepatocytes. m) No effect of insulin or GSK3β inhibitors on cyclin D1 mRNA level (n=3). n) Minimum essential medium (MEM) amino acids addition increases cyclin D1 mRNA in primary hepatocytes (one-way ANOVA with Tukey post test, n=3). o) Insulin does not change Ccnd1 mRNA in primary hepatocytes (oneway ANOVA with Tukey post test, n=3). p-q) Body, liver weights and Ccnd1 and Ccnd2 gene expression of wild-type and liver-specific cyclin D1 KO (D1 LKO) mice (combined 4 cohorts of n=3/fasting, n=5/refeeding). r) Western-blot analysis of cyclin D1 protein levels and associated signaling pathway by using nuclear and cytoplasmic liver extracts from wild-type and D1 LKO mice upon fasting (F) and 4hr refeeding (R). s) Endogenous acetylation of PGC-1α is decreased in livers of D1 LKO mice compared to wild-type mice. Western-blot analysis of acetylation of PGC-1α immunoprecipitated from liver nuclear extracts. All mice were sacrificed upon 4hr refeeding. t-u) PD 0332991 increases glycemia with similar tendency for gluconeogenic gene expression only in wild-type mice, but not in D1 LKO mice (two-tailed unpaired t-test, n=8, except n=6 for vehicle treated wild-type mice). v-w) Gluconeogenic gene expression and hepatic glucose production are increased in primary hepatocytes isolated from D1 LKO mice ( v : one-way ANOVA Tukey post test, n=3, w : two-tailed unpaired test, n=6). Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 is regulated by insulin/GSK3β and hepatic specific cyclin D1 deletion causes increased gluconeogenesis and glycemia upon refeeding. a) Cyclin D1 transcripts are increased upon refeeding. qRT-PCR analysis of Ccnd1 , Ccnd2 and Ccne1 gene expression upon overnight fasting, 4hr and 10hr refeeding in BALB/c mice livers (one-way ANOVA with Tukey post test, n=3). b) Cyclin D1 protein is increased upon refeeding. Western-blot analysis of cyclin D1 protein levels and associated signaling pathway upon fasting and refeeding measured from nuclear (N) and cytoplasmic (C) liver extracts from BALB/c mice. c) Cyclin D1-CDK4 kinase activity is increased upon 4hr refeding. In vitro 32 P incorporation into recombinant Rb by immunoprecipitated cyclin D1-CDK4 kinase from whole-cell extracts of overnight fast and 4hr refed livers. d) Western-blot analysis of cyclin D1 protein level and associated signaling pathway upon fasting and refeeding and qRT-PCR analysis of Ccnd1 and Pgc1α mRNA level in various tissues (L=liver, M=skeletal muscle, B=brown adipose tissue, W=epididymal white adipose tissue, and P=pancreas, two-tailed unpaired t-test, n=12/L, n=4/M, B, W, P). e) qRT-PCR analysis of PGC-1α target genes in liver and epididymal white adipose tissues (eWAT) upon vehicle or PD 0332991 treatment (two-tailed unpaired t-test, n=10). f) Ccnb1 and Pcna gene expressions in liver do not change upon fasting and refeeding (n=3/fast and 10hr refed, n=4/4hr refed). g) BrdU incorporation in liver does not change upon fasting and refeeding. Small intestine was used as a positive control. h) Ki-67 staining in liver does not change upon fasting and refeeding following vehicle or PD 0332991 administration. Small intestine used as a positive control. i) Ki-67 staining in liver does not change upon fasting and refeeding following GFP or cyclin D1 T286A tail-vein injection. Small intestine used as a positive control. j) Hepatic ploidy profiles of livers of GFP or cyclin D1 T286A adenovirus tail-vein injected mice do not show significant difference. Ploidy analysis of primary hepatocytes isolated from livers measured by propidium iodide staining and flow cytometry (n=6/fast and 4hr refed, n=4/10hr refed). k) Western-blot analysis of endogenous nuclear (N) and cytoplasmic (C) cyclin D1 protein level upon insulin or GSK3β inhibitors treatments in primary hepatocytes. l) PGC-1α acetylation is increased upon insulin or GSK3β inhibitors treatment in primary hepatocytes. m) No effect of insulin or GSK3β inhibitors on cyclin D1 mRNA level (n=3). n) Minimum essential medium (MEM) amino acids addition increases cyclin D1 mRNA in primary hepatocytes (one-way ANOVA with Tukey post test, n=3). o) Insulin does not change Ccnd1 mRNA in primary hepatocytes (oneway ANOVA with Tukey post test, n=3). p-q) Body, liver weights and Ccnd1 and Ccnd2 gene expression of wild-type and liver-specific cyclin D1 KO (D1 LKO) mice (combined 4 cohorts of n=3/fasting, n=5/refeeding). r) Western-blot analysis of cyclin D1 protein levels and associated signaling pathway by using nuclear and cytoplasmic liver extracts from wild-type and D1 LKO mice upon fasting (F) and 4hr refeeding (R). s) Endogenous acetylation of PGC-1α is decreased in livers of D1 LKO mice compared to wild-type mice. Western-blot analysis of acetylation of PGC-1α immunoprecipitated from liver nuclear extracts. All mice were sacrificed upon 4hr refeeding. t-u) PD 0332991 increases glycemia with similar tendency for gluconeogenic gene expression only in wild-type mice, but not in D1 LKO mice (two-tailed unpaired t-test, n=8, except n=6 for vehicle treated wild-type mice). v-w) Gluconeogenic gene expression and hepatic glucose production are increased in primary hepatocytes isolated from D1 LKO mice ( v : one-way ANOVA Tukey post test, n=3, w : two-tailed unpaired test, n=6). Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Quantitative RT-PCR, Expressing, Mouse Assay, Western Blot, Activity Assay, In Vitro, Recombinant, Immunoprecipitation, Two Tailed Test, Pyrolysis Gas Chromatography, BrdU Incorporation Assay, Positive Control, Staining, Injection, Isolation, Flow Cytometry, Cytometry

    Cyclin D1-CDK4 modulates PGC-1α acetylation through GCN5. a) Scatter plot of chemicals plotted with first test z scores on the X-axis and repeated test scores on the Y-axis. b) Fascaplysin reduces PGC-1α acetylation and Rb phoshorylation. c) Fascaplysin and PD 0332991 treatments decrease PGC-1α acetylation. d) CDK4 knockdown causes PGC-1α deacetylation. e) GCN5 knockdown blunts fascaplysin-mediated PGC-1α deacetylation. f) GCN5 acetyltransferase activity is reduced upon fascaplysin treatment (n=2, mean±S.E.M). g) Endogenous GCN5 and CDK4 interact. h) Cyclin D1-CDK4 kinase phosphorylates GCN5 in vitro. i) GCN5 T272A/S372A (AA) phosphorylation by cyclin D1-CDK4 kinase is diminished compared to GCN5 wild-type (WT). j) GCN5 T272A/S372A displays decreased acetyltransferase capacity. k) GCN5 T272A/S372A has decreased acetyltransferase activity and insensitivity to fascaplysin treatment. Kinetic constants were calculated by Michaelis-Menten equation (n=4, AU/min=arbitrary unit/min, mean±S.E.M). U-2OS cells were used for these experiments.

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: Cyclin D1-CDK4 modulates PGC-1α acetylation through GCN5. a) Scatter plot of chemicals plotted with first test z scores on the X-axis and repeated test scores on the Y-axis. b) Fascaplysin reduces PGC-1α acetylation and Rb phoshorylation. c) Fascaplysin and PD 0332991 treatments decrease PGC-1α acetylation. d) CDK4 knockdown causes PGC-1α deacetylation. e) GCN5 knockdown blunts fascaplysin-mediated PGC-1α deacetylation. f) GCN5 acetyltransferase activity is reduced upon fascaplysin treatment (n=2, mean±S.E.M). g) Endogenous GCN5 and CDK4 interact. h) Cyclin D1-CDK4 kinase phosphorylates GCN5 in vitro. i) GCN5 T272A/S372A (AA) phosphorylation by cyclin D1-CDK4 kinase is diminished compared to GCN5 wild-type (WT). j) GCN5 T272A/S372A displays decreased acetyltransferase capacity. k) GCN5 T272A/S372A has decreased acetyltransferase activity and insensitivity to fascaplysin treatment. Kinetic constants were calculated by Michaelis-Menten equation (n=4, AU/min=arbitrary unit/min, mean±S.E.M). U-2OS cells were used for these experiments.

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Pyrolysis Gas Chromatography, Activity Assay, In Vitro

    In diabetic hyperinsulinemic mice, cyclin D1-CDK4 is dysregulated and hyperactivation of cyclin D1-CDK4 attenuates the diabetic phenotype. a) qRT-PCR analysis of gluconeogenic and Ccnd1 gene expression changes upon fasting and refeeding in Lepr db/+ ( db /+) and Lepr db/db ( db/db ) mice livers (two-tailed unpaired t-test, n=3). b) Cyclin D1 protein is chronically elevated upon fasting and refeeding in livers of high fat diet fed mice compared to control diet fed mice. Nuclear (N) and cytoplasmic (C) liver extracts were used. c-d) Phosphorylation of GCN5 is elevated upon fasting in db/db or high fat diet fed (HFD) mice liver compared its respective control mice and it remains insensitive to fast-refed transitions. Western-blot analysis of GCN5 immnunoprecipiated by anti-phosphoS*P (pS*P) antibody using liver extracts from mice that were tail-vein injected with adenoviruses expressing GFP or GCN5 (F=16hr fast, R=4hr refed). e) Cyclin D1 and Rb phosphorylation levels in livers from db/db mice tail-vein injected with GFP, cyclin D1 T286A, or cyclin D1 K112E adenoviruses, shown in Fig. 4b-c . Nuclear and cytoplasmic liver extracts were used. f-g) Cyclin D1 T286A overexpression reduces gluconeogenic genes and glycemia in high fat diet fed mice (two-tailed unpaired t-test, n=6/GFP, n=7/D1 T286A). h) Cyclin D1 and Rb phosphorylation levels in livers of high fat diet fed mice that were tail-vein injected with adenoviruses expressing GFP or cyclin D1 T286A, shown in Extended Data Fig. 4f-g . i) Overall Model. Statistical significance is represented by asterisk corresponding to * P

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: In diabetic hyperinsulinemic mice, cyclin D1-CDK4 is dysregulated and hyperactivation of cyclin D1-CDK4 attenuates the diabetic phenotype. a) qRT-PCR analysis of gluconeogenic and Ccnd1 gene expression changes upon fasting and refeeding in Lepr db/+ ( db /+) and Lepr db/db ( db/db ) mice livers (two-tailed unpaired t-test, n=3). b) Cyclin D1 protein is chronically elevated upon fasting and refeeding in livers of high fat diet fed mice compared to control diet fed mice. Nuclear (N) and cytoplasmic (C) liver extracts were used. c-d) Phosphorylation of GCN5 is elevated upon fasting in db/db or high fat diet fed (HFD) mice liver compared its respective control mice and it remains insensitive to fast-refed transitions. Western-blot analysis of GCN5 immnunoprecipiated by anti-phosphoS*P (pS*P) antibody using liver extracts from mice that were tail-vein injected with adenoviruses expressing GFP or GCN5 (F=16hr fast, R=4hr refed). e) Cyclin D1 and Rb phosphorylation levels in livers from db/db mice tail-vein injected with GFP, cyclin D1 T286A, or cyclin D1 K112E adenoviruses, shown in Fig. 4b-c . Nuclear and cytoplasmic liver extracts were used. f-g) Cyclin D1 T286A overexpression reduces gluconeogenic genes and glycemia in high fat diet fed mice (two-tailed unpaired t-test, n=6/GFP, n=7/D1 T286A). h) Cyclin D1 and Rb phosphorylation levels in livers of high fat diet fed mice that were tail-vein injected with adenoviruses expressing GFP or cyclin D1 T286A, shown in Extended Data Fig. 4f-g . i) Overall Model. Statistical significance is represented by asterisk corresponding to * P

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Mouse Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Injection, Over Expression

    PD 0332991 administration or cyclin D1 T286A adenoviral overexpression does not cause toxicity compared to its respective control treatment. a) Basal physiological indexes of mice challenged with either vehicle or PD 0332991 administration (n=5). b) Basal physiological indexes of mice injected with either GFP or cyclin D1 T286A adenoviruses (n=5). (ALT=alanine transaminase, AST=aspartate transaminase, LDH=lactate dehydrogenase, mean±S.E.M)

    Journal: Nature

    Article Title: Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    doi: 10.1038/nature13267

    Figure Lengend Snippet: PD 0332991 administration or cyclin D1 T286A adenoviral overexpression does not cause toxicity compared to its respective control treatment. a) Basal physiological indexes of mice challenged with either vehicle or PD 0332991 administration (n=5). b) Basal physiological indexes of mice injected with either GFP or cyclin D1 T286A adenoviruses (n=5). (ALT=alanine transaminase, AST=aspartate transaminase, LDH=lactate dehydrogenase, mean±S.E.M)

    Article Snippet: The following antibodies were used for western blot analysis: anti-PGC-1α (sc-13067), anti-CDK4 (sc-260), anti-Cyclin D1 (sc-450) from Santa Cruz Biotechnology; anti-Acetylated-Lysine (9441), anti-GCN5 (3305), anti-pSer780 Rb (3590 or 8180), anti-Rb (9313), anti-Cyclin D1 (2978), anti-pThr308 AKT (2965), anti-pSer473 AKT (4060), anti-AKT (9272), anti-pSer9 GSK3β (9323), anti-GSK3β (9315) from Cell signaling; anti-Lamin B (ab16048), anti-Rabbit IgG (ab37415) from Abcam, anti-tubulin (05-661) from Millipore; anti-Flag (A8592), anti-βactin (A2228) from Sigma.

    Techniques: Over Expression, Mouse Assay, Injection, AST Assay

    Immunohistochemistry, α-SMA and cyclin D1 staining in sections from mice xenografted with PC3 and 22Rv1 cell lines and subjected to radiotherapy Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Upper Panel) or 22Rv1 (Lower Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 . Masson's thrichromic staining, α-SMA and cyclin D1 staining. Original Magnification 10X. Insert: original magnification 40X. (A and B Right Panel) Protein quantification. The data presented represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Immunohistochemistry, α-SMA and cyclin D1 staining in sections from mice xenografted with PC3 and 22Rv1 cell lines and subjected to radiotherapy Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Upper Panel) or 22Rv1 (Lower Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 . Masson's thrichromic staining, α-SMA and cyclin D1 staining. Original Magnification 10X. Insert: original magnification 40X. (A and B Right Panel) Protein quantification. The data presented represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: Immunohistochemistry, Staining, Mouse Assay, shRNA

    Cyclin D1staining in sections from mice xenografted with PC3 and 22Rv1 cell lines and subjected to radiotherapy Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Upper Panel) or 22Rv1 (Lower Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 : γ-H2AX, Pospho-DNA-PKCs, Ku86 and RAD51 staining and protein quantification. The data represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Cyclin D1staining in sections from mice xenografted with PC3 and 22Rv1 cell lines and subjected to radiotherapy Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Upper Panel) or 22Rv1 (Lower Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 : γ-H2AX, Pospho-DNA-PKCs, Ku86 and RAD51 staining and protein quantification. The data represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: Mouse Assay, shRNA, Staining

    Cyclin D1 promotes NHJE and HR pathways responsible of DNA-DSBs repair Schematic representation depicting the collaboration of cyclin D1 with the molecular pathways responsible for the DNA-DSBs repair. In dashed line, the molecular mechanism by which it is necessary to verify whether the action of cyclin D1 is direct or mediated by other factors.

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Cyclin D1 promotes NHJE and HR pathways responsible of DNA-DSBs repair Schematic representation depicting the collaboration of cyclin D1 with the molecular pathways responsible for the DNA-DSBs repair. In dashed line, the molecular mechanism by which it is necessary to verify whether the action of cyclin D1 is direct or mediated by other factors.

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques:

    Stable and Specific Silencing of cyclin D1 inhibits 22Rv1 onco-phenotype ( A ) Parental 22Rv1 (PRT), GFP-positive 22Rv1 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. ( B ) Cell growth assay, ( C ) cell cycle distribution by FACS and p21 waf1 , p27 KIP2 by immunoblotting, ( D ) soft agar assay and relative colony size, ( E ) invasion- and migration-assay and ( F ) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Stable and Specific Silencing of cyclin D1 inhibits 22Rv1 onco-phenotype ( A ) Parental 22Rv1 (PRT), GFP-positive 22Rv1 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. ( B ) Cell growth assay, ( C ) cell cycle distribution by FACS and p21 waf1 , p27 KIP2 by immunoblotting, ( D ) soft agar assay and relative colony size, ( E ) invasion- and migration-assay and ( F ) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: Stable Transfection, Infection, shRNA, Sequencing, FACS, Expressing, Growth Assay, Soft Agar Assay, Migration, Activation Assay, Enzyme-linked Immunosorbent Assay

    Silencing cyclin D1 radiosensitizes PC3 and 22Rv1 cell lines in vitro shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 and 22Rv1 cells were irradiated with various doses (0–8 Gy): ( A ) MTT and ( B ) (Upper Panel) colony formation assays were performed. The data presented in Figure 3A and 3B Upper Panel represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Silencing cyclin D1 radiosensitizes PC3 and 22Rv1 cell lines in vitro shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 and 22Rv1 cells were irradiated with various doses (0–8 Gy): ( A ) MTT and ( B ) (Upper Panel) colony formation assays were performed. The data presented in Figure 3A and 3B Upper Panel represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: In Vitro, shRNA, Irradiation, MTT Assay

    Silencing cyclin D1 radiosensitizersPC3 and 22Rv1 cell lines in vivo Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Left Panel) or 22Rv1 (Right Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 (T0). ( A ) Tumor volume, ( B ) tumor weights and ( C ) number of mice with tumor progression.

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Silencing cyclin D1 radiosensitizersPC3 and 22Rv1 cell lines in vivo Mice xenografted with shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 (Left Panel) or 22Rv1 (Right Panel) cells subjected to radiation treatment (5 fractions of 2 Gy were delivered over 5 consecutive days for a total dose of 10 Gy) starting when the tumor volume reached 0.5–1.0 mm 3 (T0). ( A ) Tumor volume, ( B ) tumor weights and ( C ) number of mice with tumor progression.

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: In Vivo, Mouse Assay, shRNA

    cyclin D1 depletion do not radiosentize via inducing apopotosis but rather by impairing the molecular machinery responsible of the DNA double-strand break repair shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 or 22Rv1 cells irradiated with a single dose of 4 Gy. ( A ) 6 hours post RT, H2AX activation status was investigated using and ELISA assay for γ-H2AX; the data represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: cyclin D1 depletion do not radiosentize via inducing apopotosis but rather by impairing the molecular machinery responsible of the DNA double-strand break repair shRNA-cyclin D1- (CD1) and shRNA-control-transduced (CTR) PC3 or 22Rv1 cells irradiated with a single dose of 4 Gy. ( A ) 6 hours post RT, H2AX activation status was investigated using and ELISA assay for γ-H2AX; the data represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: shRNA, Irradiation, Activation Assay, Enzyme-linked Immunosorbent Assay

    Stable and Specific Silencing of cyclin D1 inhibits PC3 onco-phenotype ( A ) Parental PC3 (PRT), GFP-positive PC3 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. ( B ) Cell growth assay, ( C ) cell cycle distribution by FACS and p21 waf1 , p27 KIP2 by immunoblotting, ( D ) soft agar assay and relative colony size, ( E ) invasion- and migration-assay and ( F ) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Journal: Oncotarget

    Article Title: Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    doi: 10.18632/oncotarget.6579

    Figure Lengend Snippet: Stable and Specific Silencing of cyclin D1 inhibits PC3 onco-phenotype ( A ) Parental PC3 (PRT), GFP-positive PC3 cells, stably infected with shRNA-cyclin D1 (CD1) vs. shRNA-control (CTR) sequence (CTR), were selected by FACS sorting and examined for cyclin D1 protein expression by immunoblotting. ( B ) Cell growth assay, ( C ) cell cycle distribution by FACS and p21 waf1 , p27 KIP2 by immunoblotting, ( D ) soft agar assay and relative colony size, ( E ) invasion- and migration-assay and ( F ) the activation status of MMP-2 and MMP-9 by ELISA assay were performed. The data presented in Figure 1B, 1C, 1D, 1E and 1F represent the mean ± SD of 3 independent experiments. Statistical analysis was performed using the Student's t -test, P

    Article Snippet: Sections were then treated with BSA (5%) for 10 minutes and finally incubated overnight with specific antibodies against cyclin D1 (H-295), RAD51 (H-92), Ku86 (H-300), α-Actin (1A4) and p-ATM (10H11.E12) all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, γH2AX (IB 100–2280) from NOVUS biologicals, used at dilutions of 1:100.

    Techniques: Stable Transfection, Infection, shRNA, Sequencing, FACS, Expressing, Growth Assay, Soft Agar Assay, Migration, Activation Assay, Enzyme-linked Immunosorbent Assay

    Maximal Sox17 expression in developing white matter coincides with OPC differentiation, and with a decline in cyclinD1 and active beta-catenin (ABC) Whole cell proteins were extracted from P5, P18 and P30 wild type C57Bl6 mouse subcortical white matter. Expression of Sox17 and some of its putative targets cyclinD1, phospho-PAK1, SFRP1, cdc42bp, MBP, b-catenin, and activated b-catenin was detected by immunoblotting using 100 µg total protein (A). Two individual brain samples for each age group are shown in this representative blot. (B). Densitometric quantitation of the Western blot after normalization with b-actin. Each bar represents a mean of 4 samples from individual brains. (C) Analysis of proteins in the Wnt/beta-catenin pathway in developing WM. Levels of Ser9 GSK3beta, P-ERK and catenin and expression of ABC, Axin2, and PCNA as detected by Western blotting with 50 ug protein. Two brain samples for each age group are shown. (D) Densitometric quantitation of Western blots after normalization with b-actin. In B and D, * P

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Sox17 regulates the Wnt/beta-catenin signaling pathway in oligodendrocyte progenitor cells

    doi: 10.1523/JNEUROSCI.3343-11.2011

    Figure Lengend Snippet: Maximal Sox17 expression in developing white matter coincides with OPC differentiation, and with a decline in cyclinD1 and active beta-catenin (ABC) Whole cell proteins were extracted from P5, P18 and P30 wild type C57Bl6 mouse subcortical white matter. Expression of Sox17 and some of its putative targets cyclinD1, phospho-PAK1, SFRP1, cdc42bp, MBP, b-catenin, and activated b-catenin was detected by immunoblotting using 100 µg total protein (A). Two individual brain samples for each age group are shown in this representative blot. (B). Densitometric quantitation of the Western blot after normalization with b-actin. Each bar represents a mean of 4 samples from individual brains. (C) Analysis of proteins in the Wnt/beta-catenin pathway in developing WM. Levels of Ser9 GSK3beta, P-ERK and catenin and expression of ABC, Axin2, and PCNA as detected by Western blotting with 50 ug protein. Two brain samples for each age group are shown. (D) Densitometric quantitation of Western blots after normalization with b-actin. In B and D, * P

    Article Snippet: The following antibodies were used in Western blot: anti-recombinant Sox17 antiserum (1:2,000; gift from Yoshiakira Kanai, Japan), mouse anti-beta catenin (1:1000; Western blotting and immunoprecipitation, 1:500 in immunocytochemistry, BD Biosciences, San Diego, CA); goat beta-catenin (1:1000, RnD Systems, Minneapolis, MN); anti-active beta-catenin (ABC, 1:1000), PLP/DM20 (1:1000), TCF4 (1:1000), anti-actin (1:5000), normal mouse IgG polyclonal (Millipore, Billerica, MA); anti-cyclinD1, p27Cip/Kip (1:800; Santa Cruz biotechnology, Inc., Santa Cruz, CA.), anti-PAK-1 (1:1000), anti-phospho-PAK1 (1:1000), anti-p44/p42 MAPK (Erk1/2) (1:1000) anti-phospho GSK3beta (1:1000 in Western blotting, 1:400 in immunocytochemistry), anti-Phospho-LRP6 (1:1000 in Western blotting, 1:500 in immunocytochemistry), anti-phospho beta-catenin (S33/37/T41) (1:1000); anti-HA (1:1000 in Western blotting, 1:200 in immunocytochemistry) (Cell Signaling Technology, Danvers, MA), anti-myelin basic protein (MBP) and anti-2’,3’ cyclic nucleotide 3’ phosphodiesterase (CNPase) (1:1000, Covance, Princeton, NJ), anti-cdc42 binding protein kinase beta (1:100), anti-C-myc (1:1000), anti-SFRP1, Axin2 (1:500; Abcam, Cambridge, MA), anti-cdc42 (1:500; Cell Biolabs, Inc, San Diego, CA).

    Techniques: Expressing, Quantitation Assay, Western Blot

    Sox17 represses cyclinD1 transcription in reporter assays Primary OPCs maintained in PDGF were transiently transfected with cyclinD1-luciferase and vector (pCMVTnT) or Sox17. Cells were allowed to recover for 48h in PDGF (A) or N1 (C) before assay. (B) Primary OPCs maintained in PDGF were transiently transfected with TOPFLASH reporter to assess TCF/b-catenin transcriptional activity. Cells were allowed to recover for 48h in PDGF. (C) Sox17 represses cyclinD1 promoter activity in the presence of co-transfected b-catenin (BCAT). In (C) and (D) ,OPCs co-transfected with TOPFLASH and beta-catenin (BCat) were allowed to recover in the absence of PDGF (N1). Equal amounts of Sox17 and beta-catenin (b-catenin) expression plasmid were co-transfected in (C) and (D). (E) Human oligodendroglioma cells (HOG) were transfected with CyclinD1-luciferase in N1 and allowed to recover in 10% FBS. (F) HOG cells transfected with TOPFLASH showed suppression of reporter activity by Sox17 similar to OPCs in (B). (G) Mean TOPFLASH/FOPFLASH luciferase activity ratios for primary OPCs transfected and maintained in PDGF as in (B). (H) TOPFLASH/FOPFLASH luciferase ratios for HOG cells transfected as in (F). * P

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Sox17 regulates the Wnt/beta-catenin signaling pathway in oligodendrocyte progenitor cells

    doi: 10.1523/JNEUROSCI.3343-11.2011

    Figure Lengend Snippet: Sox17 represses cyclinD1 transcription in reporter assays Primary OPCs maintained in PDGF were transiently transfected with cyclinD1-luciferase and vector (pCMVTnT) or Sox17. Cells were allowed to recover for 48h in PDGF (A) or N1 (C) before assay. (B) Primary OPCs maintained in PDGF were transiently transfected with TOPFLASH reporter to assess TCF/b-catenin transcriptional activity. Cells were allowed to recover for 48h in PDGF. (C) Sox17 represses cyclinD1 promoter activity in the presence of co-transfected b-catenin (BCAT). In (C) and (D) ,OPCs co-transfected with TOPFLASH and beta-catenin (BCat) were allowed to recover in the absence of PDGF (N1). Equal amounts of Sox17 and beta-catenin (b-catenin) expression plasmid were co-transfected in (C) and (D). (E) Human oligodendroglioma cells (HOG) were transfected with CyclinD1-luciferase in N1 and allowed to recover in 10% FBS. (F) HOG cells transfected with TOPFLASH showed suppression of reporter activity by Sox17 similar to OPCs in (B). (G) Mean TOPFLASH/FOPFLASH luciferase activity ratios for primary OPCs transfected and maintained in PDGF as in (B). (H) TOPFLASH/FOPFLASH luciferase ratios for HOG cells transfected as in (F). * P

    Article Snippet: The following antibodies were used in Western blot: anti-recombinant Sox17 antiserum (1:2,000; gift from Yoshiakira Kanai, Japan), mouse anti-beta catenin (1:1000; Western blotting and immunoprecipitation, 1:500 in immunocytochemistry, BD Biosciences, San Diego, CA); goat beta-catenin (1:1000, RnD Systems, Minneapolis, MN); anti-active beta-catenin (ABC, 1:1000), PLP/DM20 (1:1000), TCF4 (1:1000), anti-actin (1:5000), normal mouse IgG polyclonal (Millipore, Billerica, MA); anti-cyclinD1, p27Cip/Kip (1:800; Santa Cruz biotechnology, Inc., Santa Cruz, CA.), anti-PAK-1 (1:1000), anti-phospho-PAK1 (1:1000), anti-p44/p42 MAPK (Erk1/2) (1:1000) anti-phospho GSK3beta (1:1000 in Western blotting, 1:400 in immunocytochemistry), anti-Phospho-LRP6 (1:1000 in Western blotting, 1:500 in immunocytochemistry), anti-phospho beta-catenin (S33/37/T41) (1:1000); anti-HA (1:1000 in Western blotting, 1:200 in immunocytochemistry) (Cell Signaling Technology, Danvers, MA), anti-myelin basic protein (MBP) and anti-2’,3’ cyclic nucleotide 3’ phosphodiesterase (CNPase) (1:1000, Covance, Princeton, NJ), anti-cdc42 binding protein kinase beta (1:100), anti-C-myc (1:1000), anti-SFRP1, Axin2 (1:500; Abcam, Cambridge, MA), anti-cdc42 (1:500; Cell Biolabs, Inc, San Diego, CA).

    Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Expressing

    PDGF stimulates the Wnt/beta-catenin pathway in OPCs (A) Western analysis of MBP suppression, cyclinD1 induction and beta-catenin activation by PDGF and Wnt3a. Primary OPCs were treated with PDGF or 130 ng/ml recombinant Wnt 3a for 2 days (2D). (B) Thymidine incorporation assay comparing proliferation of primary rat OPCs treated with 10ng/ml PDGF (PDGF) or 50ng/ml human Wnt3a. 1µCi of 3H-thymidine was added to each well and cells were cultured for a further16 hours prior to analysis. Means and SD of counts from 4 wells are shown. The results are representative of two separate experiments. * P

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Sox17 regulates the Wnt/beta-catenin signaling pathway in oligodendrocyte progenitor cells

    doi: 10.1523/JNEUROSCI.3343-11.2011

    Figure Lengend Snippet: PDGF stimulates the Wnt/beta-catenin pathway in OPCs (A) Western analysis of MBP suppression, cyclinD1 induction and beta-catenin activation by PDGF and Wnt3a. Primary OPCs were treated with PDGF or 130 ng/ml recombinant Wnt 3a for 2 days (2D). (B) Thymidine incorporation assay comparing proliferation of primary rat OPCs treated with 10ng/ml PDGF (PDGF) or 50ng/ml human Wnt3a. 1µCi of 3H-thymidine was added to each well and cells were cultured for a further16 hours prior to analysis. Means and SD of counts from 4 wells are shown. The results are representative of two separate experiments. * P

    Article Snippet: The following antibodies were used in Western blot: anti-recombinant Sox17 antiserum (1:2,000; gift from Yoshiakira Kanai, Japan), mouse anti-beta catenin (1:1000; Western blotting and immunoprecipitation, 1:500 in immunocytochemistry, BD Biosciences, San Diego, CA); goat beta-catenin (1:1000, RnD Systems, Minneapolis, MN); anti-active beta-catenin (ABC, 1:1000), PLP/DM20 (1:1000), TCF4 (1:1000), anti-actin (1:5000), normal mouse IgG polyclonal (Millipore, Billerica, MA); anti-cyclinD1, p27Cip/Kip (1:800; Santa Cruz biotechnology, Inc., Santa Cruz, CA.), anti-PAK-1 (1:1000), anti-phospho-PAK1 (1:1000), anti-p44/p42 MAPK (Erk1/2) (1:1000) anti-phospho GSK3beta (1:1000 in Western blotting, 1:400 in immunocytochemistry), anti-Phospho-LRP6 (1:1000 in Western blotting, 1:500 in immunocytochemistry), anti-phospho beta-catenin (S33/37/T41) (1:1000); anti-HA (1:1000 in Western blotting, 1:200 in immunocytochemistry) (Cell Signaling Technology, Danvers, MA), anti-myelin basic protein (MBP) and anti-2’,3’ cyclic nucleotide 3’ phosphodiesterase (CNPase) (1:1000, Covance, Princeton, NJ), anti-cdc42 binding protein kinase beta (1:100), anti-C-myc (1:1000), anti-SFRP1, Axin2 (1:500; Abcam, Cambridge, MA), anti-cdc42 (1:500; Cell Biolabs, Inc, San Diego, CA).

    Techniques: Western Blot, Activation Assay, Recombinant, Thymidine Incorporation Assay, Cell Culture

    Regulation of Sox17, CyclinD1, and MBP expression in proliferating OPCs in culture (A) Representative Western blot comparing Sox17, cyclinD1, and MBP expression and regulation in OPCs maintained in PDGF (P; 10 ng/ml) for 2– 5 d. (B). Levels of expression of Sox17, cyclinD1, and MBP with time in culture (days) after densitometric analysis of Western blots and normalization with b-actin values. * P

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Sox17 regulates the Wnt/beta-catenin signaling pathway in oligodendrocyte progenitor cells

    doi: 10.1523/JNEUROSCI.3343-11.2011

    Figure Lengend Snippet: Regulation of Sox17, CyclinD1, and MBP expression in proliferating OPCs in culture (A) Representative Western blot comparing Sox17, cyclinD1, and MBP expression and regulation in OPCs maintained in PDGF (P; 10 ng/ml) for 2– 5 d. (B). Levels of expression of Sox17, cyclinD1, and MBP with time in culture (days) after densitometric analysis of Western blots and normalization with b-actin values. * P

    Article Snippet: The following antibodies were used in Western blot: anti-recombinant Sox17 antiserum (1:2,000; gift from Yoshiakira Kanai, Japan), mouse anti-beta catenin (1:1000; Western blotting and immunoprecipitation, 1:500 in immunocytochemistry, BD Biosciences, San Diego, CA); goat beta-catenin (1:1000, RnD Systems, Minneapolis, MN); anti-active beta-catenin (ABC, 1:1000), PLP/DM20 (1:1000), TCF4 (1:1000), anti-actin (1:5000), normal mouse IgG polyclonal (Millipore, Billerica, MA); anti-cyclinD1, p27Cip/Kip (1:800; Santa Cruz biotechnology, Inc., Santa Cruz, CA.), anti-PAK-1 (1:1000), anti-phospho-PAK1 (1:1000), anti-p44/p42 MAPK (Erk1/2) (1:1000) anti-phospho GSK3beta (1:1000 in Western blotting, 1:400 in immunocytochemistry), anti-Phospho-LRP6 (1:1000 in Western blotting, 1:500 in immunocytochemistry), anti-phospho beta-catenin (S33/37/T41) (1:1000); anti-HA (1:1000 in Western blotting, 1:200 in immunocytochemistry) (Cell Signaling Technology, Danvers, MA), anti-myelin basic protein (MBP) and anti-2’,3’ cyclic nucleotide 3’ phosphodiesterase (CNPase) (1:1000, Covance, Princeton, NJ), anti-cdc42 binding protein kinase beta (1:100), anti-C-myc (1:1000), anti-SFRP1, Axin2 (1:500; Abcam, Cambridge, MA), anti-cdc42 (1:500; Cell Biolabs, Inc, San Diego, CA).

    Techniques: Expressing, Western Blot

    Isoliquiritigenin regulates the members of PI3K/Akt/mTOR pathway in Ishikawa and ES-2 cells. ILQ treatment significantly reduced the expression level of p-Akt, p-mTOR, P70/S6K, and Cyclin D1 in both Ishikawa and ES-2 cells. * P

    Journal: OncoTargets and therapy

    Article Title: Effects of isoliquiritigenin on ovarian cancer cells

    doi: 10.2147/OTT.S149295

    Figure Lengend Snippet: Isoliquiritigenin regulates the members of PI3K/Akt/mTOR pathway in Ishikawa and ES-2 cells. ILQ treatment significantly reduced the expression level of p-Akt, p-mTOR, P70/S6K, and Cyclin D1 in both Ishikawa and ES-2 cells. * P

    Article Snippet: Primary antibodies, including anti-Caspas3-P17 (Cat# ab90437), anti-Bim (Cat# ab7888), anti-Bcl-2 (Cat# ab32124), anti-Bax (Cat# ab32503), anti-p-AKT (Cat# ab81283), anti-AKT (Cat# ab32505), anti-mTOR (Cat# ab2732), anti-p-mTOR (Cat# ab131538), anti-p-ERK (Cat# ab214362), anti-GSK3β (Cat# ab75745), anti-P70/S6K (Cat# ab32529), anti-Cyclin D1 (Cat# ab40754), and anti-WNT3a (Cat# ab28472), were purchased from Abcam (Cambridge, UK).

    Techniques: Expressing

    Met protects HaCaT cells against RAGE overexpression-induced cell cycle arrest. After infected with pLV-IRES-eGFP-RAGE for 48 h, HaCaT cells were treated with Met at 100 μM for 48 h. Cell cycles were measured by PI staining and flow cytometry (A). Expression of cell cycle-related proteins, including p21, Gadd45a, CyclinB1, CyclinD1, and CDK4, was measured by Western blot (B). # P

    Journal: American Journal of Translational Research

    Article Title: Receptor for advanced glycation end as drug targets in diabetes-induced skin lesion

    doi:

    Figure Lengend Snippet: Met protects HaCaT cells against RAGE overexpression-induced cell cycle arrest. After infected with pLV-IRES-eGFP-RAGE for 48 h, HaCaT cells were treated with Met at 100 μM for 48 h. Cell cycles were measured by PI staining and flow cytometry (A). Expression of cell cycle-related proteins, including p21, Gadd45a, CyclinB1, CyclinD1, and CDK4, was measured by Western blot (B). # P

    Article Snippet: Then the blots were incubated with primary antibodies specific to p21 (1:500, Abcam, Cambridge, MA, USA), Gadd45a (1:500, Abcam), CyclinB1 (1:1000, Abcam), CyclinD1 (1:2000, Cell Signaling Technology [CST], Inc., Beverley, MA, USA), CDK4 (1:2000, CST), p-p53 (1:1000, Abcam), p53 (1:1000, Abcam), caspase-3 (1:1000, CST), Bax (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-2 (1:200, Santa Cruz), p-p38 (1:1000, CST), p38 (1:1000, CST), p-NF-κBp65 (1:1000, CST), NF-κBp65 (1:1000, CST), TNF-α (1:5000, Abcam), IL-1β (1:500, Santa Cruz), IL-6 (1:100, Abcam), ICAM-1 (1:200, Abcam), COX-2 (1:500, Abcam), and GAPDH (1:1500, CST) overnight with gentle agitation at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime) for 1 h at 37°C.

    Techniques: Over Expression, Infection, Staining, Flow Cytometry, Cytometry, Expressing, Western Blot

    Effect of Met treatment on cell cycle and apoptosis in diabetic rats. After intraperitoneal injection with STZ (n=6) for 72 h, rats were treated with Met at 100 μM. Expression of cell cycle-related protein (A), including p21, Gadd45a, CyclinB1, CyclinD1, and CDK4, cell apoptosis-related protein (B, C), including p-p53, p53, caspase-3, Bax and Bcl-2, p-p38, p38, p-NF-κBP65, and NF-κBP65 was measured by western blot. ### P

    Journal: American Journal of Translational Research

    Article Title: Receptor for advanced glycation end as drug targets in diabetes-induced skin lesion

    doi:

    Figure Lengend Snippet: Effect of Met treatment on cell cycle and apoptosis in diabetic rats. After intraperitoneal injection with STZ (n=6) for 72 h, rats were treated with Met at 100 μM. Expression of cell cycle-related protein (A), including p21, Gadd45a, CyclinB1, CyclinD1, and CDK4, cell apoptosis-related protein (B, C), including p-p53, p53, caspase-3, Bax and Bcl-2, p-p38, p38, p-NF-κBP65, and NF-κBP65 was measured by western blot. ### P

    Article Snippet: Then the blots were incubated with primary antibodies specific to p21 (1:500, Abcam, Cambridge, MA, USA), Gadd45a (1:500, Abcam), CyclinB1 (1:1000, Abcam), CyclinD1 (1:2000, Cell Signaling Technology [CST], Inc., Beverley, MA, USA), CDK4 (1:2000, CST), p-p53 (1:1000, Abcam), p53 (1:1000, Abcam), caspase-3 (1:1000, CST), Bax (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-2 (1:200, Santa Cruz), p-p38 (1:1000, CST), p38 (1:1000, CST), p-NF-κBp65 (1:1000, CST), NF-κBp65 (1:1000, CST), TNF-α (1:5000, Abcam), IL-1β (1:500, Santa Cruz), IL-6 (1:100, Abcam), ICAM-1 (1:200, Abcam), COX-2 (1:500, Abcam), and GAPDH (1:1500, CST) overnight with gentle agitation at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime) for 1 h at 37°C.

    Techniques: Injection, Expressing, Western Blot

    Western blot analysis for Stat 3, Stat 3P, cyclin D1, and anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2 in human MM cell lines. Each lane contains 60 μg of protein extract from the following cell lines: lane 1 , Granta 519; lane 2 , KMS-20; lane 3 , U266; lane 4 , KMS-18; lane 5 , KMS-5; lane 6 , KMS-11; lane 7 , KMM1; lane 8 , KMS-12; lane 9 , OPM2. Lane 1 (Granta 519, mantle cell lymphoma cell line) and lane 8 (KMS12) represent the t(11;14) translocated cell lines. Expression of cyclin D1 is limited to these two cell lines. Lane 3 (U266) represents the cell line with known constitutive activation of Stat 3. Western blot analysis with the N-terminal anti-Stat 3 antibody demonstrates both Stat 3α and Stat 3β (92 and 83 kd, respectively) in all cell lines. In contrast only KMS20 and U266 show strong positivity for Stat 3 phosphorylated. Bcl-xL and Mcl-1 expression is present in all cell lines with no apparent correlation between these proteins and the presence of phosphorylated Stat 3. Bcl-2 is negative in three of the cell lines (KMS20, KMS5, and KMM1). Note that the two cell lines with expression of cyclin D1 show high levels of Bcl-2.

    Journal: The American Journal of Pathology

    Article Title: Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma

    doi:

    Figure Lengend Snippet: Western blot analysis for Stat 3, Stat 3P, cyclin D1, and anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2 in human MM cell lines. Each lane contains 60 μg of protein extract from the following cell lines: lane 1 , Granta 519; lane 2 , KMS-20; lane 3 , U266; lane 4 , KMS-18; lane 5 , KMS-5; lane 6 , KMS-11; lane 7 , KMM1; lane 8 , KMS-12; lane 9 , OPM2. Lane 1 (Granta 519, mantle cell lymphoma cell line) and lane 8 (KMS12) represent the t(11;14) translocated cell lines. Expression of cyclin D1 is limited to these two cell lines. Lane 3 (U266) represents the cell line with known constitutive activation of Stat 3. Western blot analysis with the N-terminal anti-Stat 3 antibody demonstrates both Stat 3α and Stat 3β (92 and 83 kd, respectively) in all cell lines. In contrast only KMS20 and U266 show strong positivity for Stat 3 phosphorylated. Bcl-xL and Mcl-1 expression is present in all cell lines with no apparent correlation between these proteins and the presence of phosphorylated Stat 3. Bcl-2 is negative in three of the cell lines (KMS20, KMS5, and KMM1). Note that the two cell lines with expression of cyclin D1 show high levels of Bcl-2.

    Article Snippet: In contrast to epithelial cells, plasma cells do not normally express cyclin D1, and the molecular consequences of aberrant expression of cyclin D1 in these cells may be different.

    Techniques: Western Blot, Expressing, Activation Assay

    Immunohistochemical analysis of cyclin D1 in primary MM cases. A: The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++) (case 29, group 2). B: MM with lymphoplasmacytoid features (case 32, group 2). The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++). C: MM case with nuclear positivity in 20 to 50% of tumor cells (++) and co-expression of Stat 3P (case 20, group 1). D: MM with nuclear positivity in 20 to 50% of tumor cells (++) (case 35, group 2). Note that the intensity of the staining varies from cell to cell. E: MM with nuclear positivity in 10 to 20% of tumor cells (+) and expression of Stat 3P (case 19, group 1). F: MM negative for cyclin D1. Note the presence of rare, positive plasma cells ( arrows ) (case 46, group 3). Immunoperoxidase; original magnifications: ×200 ( B ); ×400 ( A , C–F ).

    Journal: The American Journal of Pathology

    Article Title: Analysis of Signal Transducer and Activator of Transcription 3 (Stat 3) Pathway in Multiple Myeloma

    doi:

    Figure Lengend Snippet: Immunohistochemical analysis of cyclin D1 in primary MM cases. A: The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++) (case 29, group 2). B: MM with lymphoplasmacytoid features (case 32, group 2). The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++). C: MM case with nuclear positivity in 20 to 50% of tumor cells (++) and co-expression of Stat 3P (case 20, group 1). D: MM with nuclear positivity in 20 to 50% of tumor cells (++) (case 35, group 2). Note that the intensity of the staining varies from cell to cell. E: MM with nuclear positivity in 10 to 20% of tumor cells (+) and expression of Stat 3P (case 19, group 1). F: MM negative for cyclin D1. Note the presence of rare, positive plasma cells ( arrows ) (case 46, group 3). Immunoperoxidase; original magnifications: ×200 ( B ); ×400 ( A , C–F ).

    Article Snippet: In contrast to epithelial cells, plasma cells do not normally express cyclin D1, and the molecular consequences of aberrant expression of cyclin D1 in these cells may be different.

    Techniques: Immunohistochemistry, Expressing, Staining

    Effect of microinjection of antisense p27-encoding plasmid into quiescent cells on S-phase entry. (A) Vector plasmids, pcDNA3 and pCMV, or plasmids encoding either antisense p27 (pCMV5-asp27) or CDK4 (pCMV-CDK4 together with a plasmid encoding GFP [pcDNA3-GFP]) were microinjected into serum-starved NIH 3T3 cells (Parental) or their cyclin D1 derivative. BrdU was added to the cultures, and cells were subsequently stained for incorporated BrdU as described in Materials and Methods. The percentages of nuclei staining positive for both BrdU and GFP are shown (filled bars). Also shown is the percent BrdU incorporation for uninjected cells (open bars). Shown are the means plus standard errors for three independent experiments. (B) Same as panel A except that two clonal derivatives of the NIH 3T3-cyclin D1 lines, A2 and C3, were used. The results are for one experiment.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Effect of microinjection of antisense p27-encoding plasmid into quiescent cells on S-phase entry. (A) Vector plasmids, pcDNA3 and pCMV, or plasmids encoding either antisense p27 (pCMV5-asp27) or CDK4 (pCMV-CDK4 together with a plasmid encoding GFP [pcDNA3-GFP]) were microinjected into serum-starved NIH 3T3 cells (Parental) or their cyclin D1 derivative. BrdU was added to the cultures, and cells were subsequently stained for incorporated BrdU as described in Materials and Methods. The percentages of nuclei staining positive for both BrdU and GFP are shown (filled bars). Also shown is the percent BrdU incorporation for uninjected cells (open bars). Shown are the means plus standard errors for three independent experiments. (B) Same as panel A except that two clonal derivatives of the NIH 3T3-cyclin D1 lines, A2 and C3, were used. The results are for one experiment.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Plasmid Preparation, Staining, BrdU Incorporation Assay

    CDK4 kinase activity during G 1 . Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were stimulated to reenter the cell cycle by serum addition. At the indicated times, lysates were prepared and normalized for protein content, and immunoprecipitations for CDK4 were performed. Immune complexes were then assayed for CDK4-associated kinase activity with glutathione S -transferase–Rb as substrate. Cycling asynchronous cultures (designated A) were also assayed. Control immunoprecipitations from asynchronous cultures with normal rabbit serum (N) are shown. The results are representative of at least three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: CDK4 kinase activity during G 1 . Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were stimulated to reenter the cell cycle by serum addition. At the indicated times, lysates were prepared and normalized for protein content, and immunoprecipitations for CDK4 were performed. Immune complexes were then assayed for CDK4-associated kinase activity with glutathione S -transferase–Rb as substrate. Cycling asynchronous cultures (designated A) were also assayed. Control immunoprecipitations from asynchronous cultures with normal rabbit serum (N) are shown. The results are representative of at least three independent experiments.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Activity Assay

    Expression of activated MEK1 (MEK-EE) and cyclin D1 in NIH 3T3 cells. (A) Parental NIH 3T3 cells and their cyclin D1 and MEK-EE derivatives were rendered quiescent by serum starvation or maintained in a cycling asynchronous state. Under these two conditions, the levels of endogenous and exogenous MEK1, cyclin D1, and CDK4 were determined by Western blot analysis of whole-cell lysates. (B) Same as panel A, except that the cell cycle distribution of the cells was monitored by fluorescence-activated cell sorting analysis. (C) Lysates were prepared from serum-starved and asynchronous cultures of parental NIH 3T3 cells and the cyclin D1 and MEK-EE derivatives. Immunoprecipitation (IP) with antibody to CDK4 were performed. Immune complexes were assayed for CDK4-associated kinase activity with recombinant glutathione S -transferase (GST)–Rb as substrate. NRS, normal rabbit serum.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Expression of activated MEK1 (MEK-EE) and cyclin D1 in NIH 3T3 cells. (A) Parental NIH 3T3 cells and their cyclin D1 and MEK-EE derivatives were rendered quiescent by serum starvation or maintained in a cycling asynchronous state. Under these two conditions, the levels of endogenous and exogenous MEK1, cyclin D1, and CDK4 were determined by Western blot analysis of whole-cell lysates. (B) Same as panel A, except that the cell cycle distribution of the cells was monitored by fluorescence-activated cell sorting analysis. (C) Lysates were prepared from serum-starved and asynchronous cultures of parental NIH 3T3 cells and the cyclin D1 and MEK-EE derivatives. Immunoprecipitation (IP) with antibody to CDK4 were performed. Immune complexes were assayed for CDK4-associated kinase activity with recombinant glutathione S -transferase (GST)–Rb as substrate. NRS, normal rabbit serum.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Expressing, Western Blot, Fluorescence, FACS, Immunoprecipitation, Activity Assay, Recombinant

    Relative ability of stable expression of cyclin D1 or MEK-EE to reverse the cell cycle arrest induced by Ras N17 . (A) The indicated cell lines were transfected with pMT-ΔBam (control plasmid; similar results were obtained with pcDNA3 and pCMV), pMT-Ras N17 , pCMV-CDK4, pRc/CMV-CDK4 K35M (catalytically inactive CDK4), or pcDNA3-p16 together with a plasmid encoding the CD20 cell surface marker. The transfected population was identified by staining with fluorescein isothiocyanate-conjugated anti-CD20 antibody, and DNA content (2N, 4N) was monitored by staining with propidium iodide. The cell cycle distribution of the transfected population was determined by two-color flow cytometry. The cells were cultured in DMEM–5% BCS throughout the experiment. The absolute changes in the percentages of cells in G 1 compared to control transfections are shown with the mean standard error from at least three independent experiments. In control transfected cultures, the G 1 population was approximately 40%. WT, wild type. (B) Same as panel A except that Rb −/− 3T3 cells, cultured in DMEM–10% fetal bovine serum, were used. The expression plasmid for Rb was pCMV-Rb. The absolute changes in the percentages of cells in G 1 compared to control transfections are shown with the mean standard error from at least three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Relative ability of stable expression of cyclin D1 or MEK-EE to reverse the cell cycle arrest induced by Ras N17 . (A) The indicated cell lines were transfected with pMT-ΔBam (control plasmid; similar results were obtained with pcDNA3 and pCMV), pMT-Ras N17 , pCMV-CDK4, pRc/CMV-CDK4 K35M (catalytically inactive CDK4), or pcDNA3-p16 together with a plasmid encoding the CD20 cell surface marker. The transfected population was identified by staining with fluorescein isothiocyanate-conjugated anti-CD20 antibody, and DNA content (2N, 4N) was monitored by staining with propidium iodide. The cell cycle distribution of the transfected population was determined by two-color flow cytometry. The cells were cultured in DMEM–5% BCS throughout the experiment. The absolute changes in the percentages of cells in G 1 compared to control transfections are shown with the mean standard error from at least three independent experiments. In control transfected cultures, the G 1 population was approximately 40%. WT, wild type. (B) Same as panel A except that Rb −/− 3T3 cells, cultured in DMEM–10% fetal bovine serum, were used. The expression plasmid for Rb was pCMV-Rb. The absolute changes in the percentages of cells in G 1 compared to control transfections are shown with the mean standard error from at least three independent experiments.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Expressing, Transfection, Plasmid Preparation, Marker, Staining, Flow Cytometry, Cytometry, Cell Culture

    Effect of Ras N17 expression on CDK4 activity and D cyclin levels. (A) NIH 3T3 cells, cultured in DMEM–5% BCS, were transfected with plasmids encoding the indicated proteins or vector alone together with a plasmid encoding the surface marker CD20. Forty-two hours later, transfected cells were isolated by magnetic sorting with Dynabeads (see Materials and Methods). Extracts were prepared, normalized for protein content, and subjected to immunoprecipitation for CDK4. CDK4-associated kinase was measured by using glutathione S -transferase (GST)–Rb as a substrate. In lane 1, control normal rabbit serum was used. Lane 2 is the control (pMT-ΔBam, vector-alone transfection; the same results were obtained when pcDNA3 was used as the vector control) set to 100% kinase activity. The bar graph represents the relative kinase activity with the mean standard error from at least three independent experiments. A representative autoradiogram is shown. (B) Same as panel A except that cell extracts were used for Western blot analysis for cyclin D1, cyclin D2, cyclin D3, and CDK4. Indicated proteins were detected by enhanced chemiluminescence.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Effect of Ras N17 expression on CDK4 activity and D cyclin levels. (A) NIH 3T3 cells, cultured in DMEM–5% BCS, were transfected with plasmids encoding the indicated proteins or vector alone together with a plasmid encoding the surface marker CD20. Forty-two hours later, transfected cells were isolated by magnetic sorting with Dynabeads (see Materials and Methods). Extracts were prepared, normalized for protein content, and subjected to immunoprecipitation for CDK4. CDK4-associated kinase was measured by using glutathione S -transferase (GST)–Rb as a substrate. In lane 1, control normal rabbit serum was used. Lane 2 is the control (pMT-ΔBam, vector-alone transfection; the same results were obtained when pcDNA3 was used as the vector control) set to 100% kinase activity. The bar graph represents the relative kinase activity with the mean standard error from at least three independent experiments. A representative autoradiogram is shown. (B) Same as panel A except that cell extracts were used for Western blot analysis for cyclin D1, cyclin D2, cyclin D3, and CDK4. Indicated proteins were detected by enhanced chemiluminescence.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Expressing, Activity Assay, Cell Culture, Transfection, Plasmid Preparation, Marker, Isolation, Immunoprecipitation, Western Blot

    Association of p27 with cyclin D1 and CDK4. (A) Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were then restimulated by serum addition. At the indicated times, lysates were prepared and immunoprecipitations (IP) for p27 were performed. Immune complexes were resolved in a denaturing gel, and Western blot analysis was performed for p27. Lysates prepared from cycling asynchronous cultures (designated A) were also analyzed. (B, C, D, and E) Same as panel A except that antibodies used for immunoprecipitation and Western blot analysis (Blot) were as indicated. The experiments for each of the blots shown in panels A, B, C, D, and E were performed in parallel. Indicated proteins were detected by enhanced chemiluminescence, and the exposure time in each of the panels is the same; thus, a comparison of the relative amounts of p27 associated with cyclin D1 and CDK4 can be made. The results are representative of at least five independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Association of p27 with cyclin D1 and CDK4. (A) Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were then restimulated by serum addition. At the indicated times, lysates were prepared and immunoprecipitations (IP) for p27 were performed. Immune complexes were resolved in a denaturing gel, and Western blot analysis was performed for p27. Lysates prepared from cycling asynchronous cultures (designated A) were also analyzed. (B, C, D, and E) Same as panel A except that antibodies used for immunoprecipitation and Western blot analysis (Blot) were as indicated. The experiments for each of the blots shown in panels A, B, C, D, and E were performed in parallel. Indicated proteins were detected by enhanced chemiluminescence, and the exposure time in each of the panels is the same; thus, a comparison of the relative amounts of p27 associated with cyclin D1 and CDK4 can be made. The results are representative of at least five independent experiments.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Western Blot, Immunoprecipitation

    Association between cyclin D1 and CDK4. (A) Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were then restimulated by serum addition. At the indicated times, lysates were prepared and immunoprecipitations (IP) for cyclin D1 were performed. Immune complexes were resolved in a denaturing gel, and Western blot analysis was performed for cyclin D1. Lysates prepared from cycling asynchronous cultures (designated A) were also analyzed. (B) Same as panel A except that CDK4 was analyzed. (C and D) Same as above except that CDK4 immunoprecipitates were analyzed for associated cyclin D1 and cyclin D1 immunoprecipitates were analyzed for associated CDK4. The experiments performed for each of the blots shown in panels A, B, C, and D were performed in parallel. Indicated proteins were detected by enhanced chemiluminescence, and the exposure time in each of the panels is the same; thus, the relative amounts of cyclin D1 associated with CDK4 and vice versa can be compared. The results are representative of at least five independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

    doi:

    Figure Lengend Snippet: Association between cyclin D1 and CDK4. (A) Parental NIH 3T3 cells and their MEK-EE and cyclin D1 derivatives were rendered quiescent by serum starvation. Cells were then restimulated by serum addition. At the indicated times, lysates were prepared and immunoprecipitations (IP) for cyclin D1 were performed. Immune complexes were resolved in a denaturing gel, and Western blot analysis was performed for cyclin D1. Lysates prepared from cycling asynchronous cultures (designated A) were also analyzed. (B) Same as panel A except that CDK4 was analyzed. (C and D) Same as above except that CDK4 immunoprecipitates were analyzed for associated cyclin D1 and cyclin D1 immunoprecipitates were analyzed for associated CDK4. The experiments performed for each of the blots shown in panels A, B, C, and D were performed in parallel. Indicated proteins were detected by enhanced chemiluminescence, and the exposure time in each of the panels is the same; thus, the relative amounts of cyclin D1 associated with CDK4 and vice versa can be compared. The results are representative of at least five independent experiments.

    Article Snippet: This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1.

    Techniques: Western Blot

    In the experimental arm, overall survival (3A), disease-free survival (3B), locoregional recurrence-free survival (3C), and distant metastasis-free survival (3D) in OSCC patients with low and high cyclin D1 expression.

    Journal: Molecular cancer therapeutics

    Article Title: Elevated cyclin D1 expression is predictive for a benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease

    doi: 10.1158/1535-7163.MCT-12-1013

    Figure Lengend Snippet: In the experimental arm, overall survival (3A), disease-free survival (3B), locoregional recurrence-free survival (3C), and distant metastasis-free survival (3D) in OSCC patients with low and high cyclin D1 expression.

    Article Snippet: In brief, after deparaffinization, endogenous peroxidase was blocked, and the sections were heated by water bath at 98°C with 0.01M citrate buffer solution (pH 6.0) for 20min to retrieve antigen, and cooled at room temperature, then washed with PBS 3 times for 5min each, then incubated with the rabbit monoclonal antibody to cyclin D1 (clone-EPR2241, Epitomics, Inc., USA) at 1:150 dilution overnight at 4°C.

    Techniques: Expressing

    In the control arm, overall survival (2A), disease-free survival (2B), locoregional recurrence-free survival (2C), and distant metastasis-free survival (2D) in OSCC patients with low and high cyclin D1 expression.

    Journal: Molecular cancer therapeutics

    Article Title: Elevated cyclin D1 expression is predictive for a benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease

    doi: 10.1158/1535-7163.MCT-12-1013

    Figure Lengend Snippet: In the control arm, overall survival (2A), disease-free survival (2B), locoregional recurrence-free survival (2C), and distant metastasis-free survival (2D) in OSCC patients with low and high cyclin D1 expression.

    Article Snippet: In brief, after deparaffinization, endogenous peroxidase was blocked, and the sections were heated by water bath at 98°C with 0.01M citrate buffer solution (pH 6.0) for 20min to retrieve antigen, and cooled at room temperature, then washed with PBS 3 times for 5min each, then incubated with the rabbit monoclonal antibody to cyclin D1 (clone-EPR2241, Epitomics, Inc., USA) at 1:150 dilution overnight at 4°C.

    Techniques: Expressing

    Cyclin D1 expression, treatment, and survival outcomes

    Journal: Molecular cancer therapeutics

    Article Title: Elevated cyclin D1 expression is predictive for a benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease

    doi: 10.1158/1535-7163.MCT-12-1013

    Figure Lengend Snippet: Cyclin D1 expression, treatment, and survival outcomes

    Article Snippet: In brief, after deparaffinization, endogenous peroxidase was blocked, and the sections were heated by water bath at 98°C with 0.01M citrate buffer solution (pH 6.0) for 20min to retrieve antigen, and cooled at room temperature, then washed with PBS 3 times for 5min each, then incubated with the rabbit monoclonal antibody to cyclin D1 (clone-EPR2241, Epitomics, Inc., USA) at 1:150 dilution overnight at 4°C.

    Techniques: Expressing

    Overall survival (4A, 4B) and distant metastasis-free survival (4C, 4D) in cN2 OSCC patients in the experimental and control arms according to the low cyclin D1 expression (4A, 4C) and high cyclin D1expression (4B, 4D).

    Journal: Molecular cancer therapeutics

    Article Title: Elevated cyclin D1 expression is predictive for a benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease

    doi: 10.1158/1535-7163.MCT-12-1013

    Figure Lengend Snippet: Overall survival (4A, 4B) and distant metastasis-free survival (4C, 4D) in cN2 OSCC patients in the experimental and control arms according to the low cyclin D1 expression (4A, 4C) and high cyclin D1expression (4B, 4D).

    Article Snippet: In brief, after deparaffinization, endogenous peroxidase was blocked, and the sections were heated by water bath at 98°C with 0.01M citrate buffer solution (pH 6.0) for 20min to retrieve antigen, and cooled at room temperature, then washed with PBS 3 times for 5min each, then incubated with the rabbit monoclonal antibody to cyclin D1 (clone-EPR2241, Epitomics, Inc., USA) at 1:150 dilution overnight at 4°C.

    Techniques: Expressing

    Relative frequency distribution of cyclin D1 expression index in the 232 patients with resectable locally advanced oral squamous cell carcinoma.

    Journal: Molecular cancer therapeutics

    Article Title: Elevated cyclin D1 expression is predictive for a benefit from TPF induction chemotherapy in oral squamous cell carcinoma patients with advanced nodal disease

    doi: 10.1158/1535-7163.MCT-12-1013

    Figure Lengend Snippet: Relative frequency distribution of cyclin D1 expression index in the 232 patients with resectable locally advanced oral squamous cell carcinoma.

    Article Snippet: In brief, after deparaffinization, endogenous peroxidase was blocked, and the sections were heated by water bath at 98°C with 0.01M citrate buffer solution (pH 6.0) for 20min to retrieve antigen, and cooled at room temperature, then washed with PBS 3 times for 5min each, then incubated with the rabbit monoclonal antibody to cyclin D1 (clone-EPR2241, Epitomics, Inc., USA) at 1:150 dilution overnight at 4°C.

    Techniques: Expressing

    Effects of a dominant negative shuttling-deficient hnRNP A1 mutant on Akt-dependent cyclin D1 and c- myc IRES activity following rapamycin exposure. A , expression of the NLS-A1-HA mutant in U87 cells. Immunofluorescence microscopy of untransduced (mock-infected,

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Effects of a dominant negative shuttling-deficient hnRNP A1 mutant on Akt-dependent cyclin D1 and c- myc IRES activity following rapamycin exposure. A , expression of the NLS-A1-HA mutant in U87 cells. Immunofluorescence microscopy of untransduced (mock-infected,

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: Dominant Negative Mutation, Mutagenesis, Activity Assay, Expressing, Immunofluorescence, Microscopy, Infection

    Knockdown of hnRNP A1 alters polysome distribution of cyclin D1 and c- myc mRNAs. A , polysome distributions of cyclin D1, c- myc , and actin mRNAs in hnRNP A1 knockdown PTEN -/- and PTEN +/+ MEFs treated with or without rapamycin. Extracts were prepared

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Knockdown of hnRNP A1 alters polysome distribution of cyclin D1 and c- myc mRNAs. A , polysome distributions of cyclin D1, c- myc , and actin mRNAs in hnRNP A1 knockdown PTEN -/- and PTEN +/+ MEFs treated with or without rapamycin. Extracts were prepared

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques:

    Binding of hnRNP A1 to minimal cyclin D1 and c- myc IRESs in a yeast three-hybrid system. A, schematic diagram showing components of the three-hybrid assay. Clones encoding hnRNP A1 fused to a transcriptional activation domain bound a bifunctional hybrid

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Binding of hnRNP A1 to minimal cyclin D1 and c- myc IRESs in a yeast three-hybrid system. A, schematic diagram showing components of the three-hybrid assay. Clones encoding hnRNP A1 fused to a transcriptional activation domain bound a bifunctional hybrid

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: Binding Assay, Hybrid Assay, Clone Assay, Activation Assay

    Association of hnRNP A1 with the cyclin D1 and c- myc IRESs in vitro and in intact cells. A , RNA-EMSA of 32 P-labeled cyclin D1 or c- myc minimal IRES sequences incubated with GST-control (-,-) or GST-hnRNP A1 (-,+). Supershift was assessed by the addition

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Association of hnRNP A1 with the cyclin D1 and c- myc IRESs in vitro and in intact cells. A , RNA-EMSA of 32 P-labeled cyclin D1 or c- myc minimal IRES sequences incubated with GST-control (-,-) or GST-hnRNP A1 (-,+). Supershift was assessed by the addition

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: In Vitro, Labeling, Incubation

    Knockdown of hnRNP A1 abrogates Akt-dependent cyclin D1 and c- myc IRES activity following rapamycin exposure. U87 and U87 PTEN cells in which hnRNP A1 expression was inhibited via siRNA were transiently transfected with the indicated dicistronic reporter

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Knockdown of hnRNP A1 abrogates Akt-dependent cyclin D1 and c- myc IRES activity following rapamycin exposure. U87 and U87 PTEN cells in which hnRNP A1 expression was inhibited via siRNA were transiently transfected with the indicated dicistronic reporter

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: Activity Assay, Expressing, Transfection

    Serine 199 is differentially phosphorylated in cyclin D1 or c- myc IRES bound hnRNP A1 in an Akt-dependent manner following rapamycin exposure. Biotinylated cyclin D1 or c- myc IRES RNAs were used to pull-down hnRNP A1 from cytoplasmic extracts of

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Serine 199 is differentially phosphorylated in cyclin D1 or c- myc IRES bound hnRNP A1 in an Akt-dependent manner following rapamycin exposure. Biotinylated cyclin D1 or c- myc IRES RNAs were used to pull-down hnRNP A1 from cytoplasmic extracts of

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques:

    The 5′-UTRs of the human cyclin D1 and c- myc mRNAs contain short regions that exhibit Akt-dependent IRES activity following rapamycin exposure. A, schematic diagram showing dicistronic constructs. B , DNA segments corresponding to the indicated

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: The 5′-UTRs of the human cyclin D1 and c- myc mRNAs contain short regions that exhibit Akt-dependent IRES activity following rapamycin exposure. A, schematic diagram showing dicistronic constructs. B , DNA segments corresponding to the indicated

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: Activity Assay, Construct

    Akt negatively regulates cyclin D1 and c- myc IRES activity in vitro . A , siRNA mediated knockdown of hnRNP A1. U87 and U87 PTEN were transiently transfected with siRNAs targeting hnRNP A1 or a nontargeting scrambled ( scr ) sequence and exposed to rapamycin

    Journal:

    Article Title: Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling * Internal Ribosome Entry Site Function through Akt Signaling * S⃞

    doi: 10.1074/jbc.M801185200

    Figure Lengend Snippet: Akt negatively regulates cyclin D1 and c- myc IRES activity in vitro . A , siRNA mediated knockdown of hnRNP A1. U87 and U87 PTEN were transiently transfected with siRNAs targeting hnRNP A1 or a nontargeting scrambled ( scr ) sequence and exposed to rapamycin

    Article Snippet: Anti-GST was from MBL International Corp. (Woburn, MA); anti-hnRNP A1 was from Abcam (Cambridge, MA); anti-phospho-Akt-substrate, anti-Akt, anti-phospho-(Thr308 )-Akt, and anti-phospho-(Ser473 )-Akt were from Cell Signaling (Danvers, MA); anti-HA antibodies 3F10 and 12CA5 were from Roche Applied Science; cyclin D1 was from BD Pharmingen; c- myc (clone 9E11) was from Upstate Biotechnology; and anti-actin antibody was from Sigma.

    Techniques: Activity Assay, In Vitro, Transfection, Sequencing

    Nuclear localization of cyclin D1 is impaired in p27 −/− PMECs. (A) Western analysis of cytoplasmic and nuclear extracts from infected PMECs with antibodies against cyclin D1, p27, and PCNA. (B) Immunofluorescence analysis was used to detect cellular localization of cyclin D1 in infected PMECs. DAPI staining of nuclei is pictured directly below the corresponding cyclin D1 immunofluorescence. Magnification, ×400. Values shown represent the percentage of total nuclei that were positive for cyclin D1 staining. A total of 500 nuclei were counted per experimental condition.

    Journal: Molecular and Cellular Biology

    Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells

    doi: 10.1128/MCB.22.7.2204-2219.2002

    Figure Lengend Snippet: Nuclear localization of cyclin D1 is impaired in p27 −/− PMECs. (A) Western analysis of cytoplasmic and nuclear extracts from infected PMECs with antibodies against cyclin D1, p27, and PCNA. (B) Immunofluorescence analysis was used to detect cellular localization of cyclin D1 in infected PMECs. DAPI staining of nuclei is pictured directly below the corresponding cyclin D1 immunofluorescence. Magnification, ×400. Values shown represent the percentage of total nuclei that were positive for cyclin D1 staining. A total of 500 nuclei were counted per experimental condition.

    Article Snippet: In contrast, only 3.0, 6.2, and 18.1% of the p27−/− cells infected with pBabe- erbB2 , - cyclin D1 , or - cyclin D1 ( T286A ), respectively, demonstrated cyclin D1 in the nucleus.

    Techniques: Western Blot, Infection, Immunofluorescence, Staining

    Overexpression of ErbB2 increases cyclin D1 expression in p27 +/+ and p27 +/− PMECs but not p27 −/− PMECs. (A) Western analysis of whole-cell extracts (WCE), 9E10 immunoprecipitates (IP: 9E10), or ErbB2 immunoprecipitates (IP: ErbB2) from uninfected p27 +/+ PMECs (NI) or p27 +/+ PMECs infected with pBabe or pBabe- erbB2 . Blots were probed with antibodies against ErbB2 (left panels) or phosphotyrosine (right panel). (B) PMECs infected with empty pBabe or pBabe- erbB2 were cultured for 10 days from a single-cell suspension embedded in growth factor-reduced Matrigel. Cultures were photographed at magnifications of ×100 (panels 1 to 6) and ×400 (panels 7 to 12). The photographs shown are representative of results obtained in three independent experiments. Panels 10 to 12, whole-mount immunohistochemical detection of E-cadherin in organoid cultures. DAPI-stained nuclei are shown to the right. (C) Western analysis of cell extracts harvested from PMEC monolayers infected with pBabe, pBabe- erbB2 , pBabe- cyclin D1 , or pBabe- cyclin D1 ( T286A ). WB, primary antibodies used for Western blot analysis, listed at the left. The 9E10 antibody is against the myc epitope tag. Molecular masses are shown at right in kilodaltons. The results presented here are representative of results obtained in three independent experiments. (D) Western analysis of cell extracts harvested from PMEC organoid cultures infected with pBabe, pBabe- erbB2 , pBabe- cyclin D1 , or pBabe- cyclin D1 ( T286A ) . (E) Northern analysis of total cellular RNA harvested from infected PMECs. The cDNA probe used for hybridization is indicated (cyclin D1 and GAPDH). The positions of the 28S and 18S rRNAs are indicated.

    Journal: Molecular and Cellular Biology

    Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells

    doi: 10.1128/MCB.22.7.2204-2219.2002

    Figure Lengend Snippet: Overexpression of ErbB2 increases cyclin D1 expression in p27 +/+ and p27 +/− PMECs but not p27 −/− PMECs. (A) Western analysis of whole-cell extracts (WCE), 9E10 immunoprecipitates (IP: 9E10), or ErbB2 immunoprecipitates (IP: ErbB2) from uninfected p27 +/+ PMECs (NI) or p27 +/+ PMECs infected with pBabe or pBabe- erbB2 . Blots were probed with antibodies against ErbB2 (left panels) or phosphotyrosine (right panel). (B) PMECs infected with empty pBabe or pBabe- erbB2 were cultured for 10 days from a single-cell suspension embedded in growth factor-reduced Matrigel. Cultures were photographed at magnifications of ×100 (panels 1 to 6) and ×400 (panels 7 to 12). The photographs shown are representative of results obtained in three independent experiments. Panels 10 to 12, whole-mount immunohistochemical detection of E-cadherin in organoid cultures. DAPI-stained nuclei are shown to the right. (C) Western analysis of cell extracts harvested from PMEC monolayers infected with pBabe, pBabe- erbB2 , pBabe- cyclin D1 , or pBabe- cyclin D1 ( T286A ). WB, primary antibodies used for Western blot analysis, listed at the left. The 9E10 antibody is against the myc epitope tag. Molecular masses are shown at right in kilodaltons. The results presented here are representative of results obtained in three independent experiments. (D) Western analysis of cell extracts harvested from PMEC organoid cultures infected with pBabe, pBabe- erbB2 , pBabe- cyclin D1 , or pBabe- cyclin D1 ( T286A ) . (E) Northern analysis of total cellular RNA harvested from infected PMECs. The cDNA probe used for hybridization is indicated (cyclin D1 and GAPDH). The positions of the 28S and 18S rRNAs are indicated.

    Article Snippet: In contrast, only 3.0, 6.2, and 18.1% of the p27−/− cells infected with pBabe- erbB2 , - cyclin D1 , or - cyclin D1 ( T286A ), respectively, demonstrated cyclin D1 in the nucleus.

    Techniques: Over Expression, Expressing, Western Blot, Infection, Cell Culture, Immunohistochemistry, Staining, Northern Blot, Hybridization

    Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

    Journal: Molecular and Cellular Biology

    Article Title: ErbB2/Neu-Induced, Cyclin D1-Dependent Transformation Is Accelerated in p27-Haploinsufficient Mammary Epithelial Cells but Impaired in p27-Null Cells

    doi: 10.1128/MCB.22.7.2204-2219.2002

    Figure Lengend Snippet: Activity of Cdk4 is impaired in p27 −/− PMECs and is not restored by ErbB2 or cyclin D1 overexpression. (A) Cell extracts from infected PMECs were immunoprecipitated (IP) with an antibody against Cdk4. Immune complexes were divided in half and tested in an in vitro kinase assay with pRb as a substrate (upper panel) or used for Western blot analysis (WB) with a Cdk4 antibody (lower panel). Kinase reactions were performed in the presence of [γ- 32 P]ATP and then resolved by SDS-PAGE. (B) Whole-cell extracts from infected PMECs were subjected to Western blot analysis using the antibodies indicated at the right. (C) Whole-cell extracts from PMECs infected with pBabe- p27 or pBabe- E2F1 or from uninfected PMECs (N.I.) were immunoprecipitated with an antibody against Cdk4. Products were divided in half and used in an in vitro kinase reaction against pRb or in Western analysis for Cdk4 as described in panel A. Whole-cell extracts were used for Western analysis with p27 or E2F1 antibodies. (D) p27 −/− PMECs infected with the indicated viruses were cultured in the presence of cycloheximide (Cyclohex) (1 μg/ml). Cell extracts were analyzed for cyclin D1 expression at various time points following cycloheximide administration. (E) Whole-cell extracts from p27 −/− PMECs infected with the indicated viruses or from uninfected p27 −/− PMECs (N.I.) were used for detection of cyclin D1 by Western analysis (top panel) or were immunoprecipitated with an antibody against Cdk4 and analyzed for coprecipitation of cyclin D1.

    Article Snippet: In contrast, only 3.0, 6.2, and 18.1% of the p27−/− cells infected with pBabe- erbB2 , - cyclin D1 , or - cyclin D1 ( T286A ), respectively, demonstrated cyclin D1 in the nucleus.

    Techniques: Activity Assay, Over Expression, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Western Blot, SDS Page, Cell Culture, Expressing

    Proteome analysis of cyclin D1 conditioned medium identifies inflammatory cytokines and dendritic cell maturation pathways A. Venn diagram comparing the overlap of proteins in secretomes of hTERT-control stroma and hTERT-cyclin D1 stroma cells ( n = 3 for each condition). B. Hierarchical clustering heat map of hTERT-control stroma and hTERT-cyclin D1 stroma secretomes based on LFQ intensities of identified proteins. C. Volcano plot of proteins identified in secretome analysis. Log2 ratios of LFQ intensities in hTERT-cyclin D1 stroma vs . hTERT-control stroma were plotted against negative log10 Student’s t -test p-values. Significantly changed proteins, defined as absolute fold-change > 2 and p

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Proteome analysis of cyclin D1 conditioned medium identifies inflammatory cytokines and dendritic cell maturation pathways A. Venn diagram comparing the overlap of proteins in secretomes of hTERT-control stroma and hTERT-cyclin D1 stroma cells ( n = 3 for each condition). B. Hierarchical clustering heat map of hTERT-control stroma and hTERT-cyclin D1 stroma secretomes based on LFQ intensities of identified proteins. C. Volcano plot of proteins identified in secretome analysis. Log2 ratios of LFQ intensities in hTERT-cyclin D1 stroma vs . hTERT-control stroma were plotted against negative log10 Student’s t -test p-values. Significantly changed proteins, defined as absolute fold-change > 2 and p

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques:

    Cyclin D1 induces Osteopontin (OPN) abundance A. Western blot analysis for OPN in cyclin D1 -/- MEFs transduced with either control vector or a cyclin D1 expressing vector. β-actin is a loading control. B. Western blot analysis of either wild type or cyclin D1 -/- MEFs for the abundance of OPN and its bioactive cleaved forms, MMP3 (cleavge enzyme for OPN) and β-actin loading control. C. ELISA of OPN in cell culture media of either wild type or cyclin D1 -/- MEFs. The serum-free cell cuture media were collected after 48 hours. D. Schematic representation of transgenic mice in which a cyclin D1 cDNA was induced in the mammary gland upon addition of doxycycline. E. Immunohistochemistry for OPN in the mammary gland of transgenic mice with quantitation of IHC shown as mean + SEM for N = 3 separate mice. F. Schematic representation of transgenic paradigm to delete cyclin D1 in mice using tamoxifen inducible Cre expression. G. Immunohistochemistry for OPN in the mammary gland of transgenic mice with quantitation of IHC shown as mean ± SEM for N = 3 separate mice. H. Assay of OPN mediated induction of embryonic stem (ES) cells. Image of the plates in which ES cell assays were conducted using either vehicle or OPN, with representative colonies shown at high magnification (200x). Quantitation of ES cell colony number shown as mean ± SEM for N = 3.

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Cyclin D1 induces Osteopontin (OPN) abundance A. Western blot analysis for OPN in cyclin D1 -/- MEFs transduced with either control vector or a cyclin D1 expressing vector. β-actin is a loading control. B. Western blot analysis of either wild type or cyclin D1 -/- MEFs for the abundance of OPN and its bioactive cleaved forms, MMP3 (cleavge enzyme for OPN) and β-actin loading control. C. ELISA of OPN in cell culture media of either wild type or cyclin D1 -/- MEFs. The serum-free cell cuture media were collected after 48 hours. D. Schematic representation of transgenic mice in which a cyclin D1 cDNA was induced in the mammary gland upon addition of doxycycline. E. Immunohistochemistry for OPN in the mammary gland of transgenic mice with quantitation of IHC shown as mean + SEM for N = 3 separate mice. F. Schematic representation of transgenic paradigm to delete cyclin D1 in mice using tamoxifen inducible Cre expression. G. Immunohistochemistry for OPN in the mammary gland of transgenic mice with quantitation of IHC shown as mean ± SEM for N = 3 separate mice. H. Assay of OPN mediated induction of embryonic stem (ES) cells. Image of the plates in which ES cell assays were conducted using either vehicle or OPN, with representative colonies shown at high magnification (200x). Quantitation of ES cell colony number shown as mean ± SEM for N = 3.

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Western Blot, Transduction, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Transgenic Assay, Mouse Assay, Immunohistochemistry, Quantitation Assay

    Stromal cyclin D1 expression increases breast tumor growth in mice A. Mice were injected with either hTERT control (control stroma ) or hTERT-cyclin D1 (cyclin D1 stroma ), in equal number together with MDA-MB-231 cells (RFP) and the tumor size assessed with B. tumor weight and C. tumor volume shown as mean ± SEM for N = 6 separate animals in each group. D. Immunohistochemical analysis of the tumors for cell proliferation (Ki-67), E. apoptosis (TUNEL), F. α-SMA and G. calponin with quantitation shown as mean ± SEM for N = 6 separate animals in each group.

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Stromal cyclin D1 expression increases breast tumor growth in mice A. Mice were injected with either hTERT control (control stroma ) or hTERT-cyclin D1 (cyclin D1 stroma ), in equal number together with MDA-MB-231 cells (RFP) and the tumor size assessed with B. tumor weight and C. tumor volume shown as mean ± SEM for N = 6 separate animals in each group. D. Immunohistochemical analysis of the tumors for cell proliferation (Ki-67), E. apoptosis (TUNEL), F. α-SMA and G. calponin with quantitation shown as mean ± SEM for N = 6 separate animals in each group.

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Expressing, Mouse Assay, Injection, Multiple Displacement Amplification, Immunohistochemistry, TUNEL Assay, Quantitation Assay

    Cyclin D1 stroma expression increases breast cancer inflammation A. Immunohistochemical analysis of the tumors for A. - C. mitophagy/autophagy (LC3B, BECN-1, LAMP-1), D. angiogenesis (Von Willebrand Factor (VWF), E. F4/80 + macrophages, F. CD11b + macrophages, with quantitation shown as mean ± SEM for N = 6 separate animals in each group.

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Cyclin D1 stroma expression increases breast cancer inflammation A. Immunohistochemical analysis of the tumors for A. - C. mitophagy/autophagy (LC3B, BECN-1, LAMP-1), D. angiogenesis (Von Willebrand Factor (VWF), E. F4/80 + macrophages, F. CD11b + macrophages, with quantitation shown as mean ± SEM for N = 6 separate animals in each group.

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Expressing, Immunohistochemistry, Quantitation Assay

    Fibroblast cyclin D1 expression increases secretion of inflammatory cytokines A. Soluble growth factor/cytokine/receptor array of conditioned medium from hTERT-control stroma or hTERT-cyclin D1 stroma or C. - F. , from cyclin D1 +/+ or cyclin D1 -/- MEFs. Data is shown as fold change.

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Fibroblast cyclin D1 expression increases secretion of inflammatory cytokines A. Soluble growth factor/cytokine/receptor array of conditioned medium from hTERT-control stroma or hTERT-cyclin D1 stroma or C. - F. , from cyclin D1 +/+ or cyclin D1 -/- MEFs. Data is shown as fold change.

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Expressing

    Cyclin D1 conditioned medium induces expansion of CD34 positive hematopoietic stem cells (HSCs) and promotes differentiation of CD34 positive hematopoietic stem cells (HSCs) into dendritic cells A. The bone marrow cells were cultured in media derived from cyclin D1 -/- or cyclin D1 +/+ MEFs for 9 days and analyzed for CD34 expression by flow cytometry as described in Materials and Methods. Histogram represents the percentage of CD34 + cells. The gate was set based on unstained and/or isotype control for each sample. B. - C. The supernatant from cyclin D1 -/- or cyclin D1 +/+ MEFs were incubated with ES cells and assessed for proliferation or D. , E. colony number. Quantitation of ES cell colony number shown as mean ± SEM for N = 3.

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Cyclin D1 conditioned medium induces expansion of CD34 positive hematopoietic stem cells (HSCs) and promotes differentiation of CD34 positive hematopoietic stem cells (HSCs) into dendritic cells A. The bone marrow cells were cultured in media derived from cyclin D1 -/- or cyclin D1 +/+ MEFs for 9 days and analyzed for CD34 expression by flow cytometry as described in Materials and Methods. Histogram represents the percentage of CD34 + cells. The gate was set based on unstained and/or isotype control for each sample. B. - C. The supernatant from cyclin D1 -/- or cyclin D1 +/+ MEFs were incubated with ES cells and assessed for proliferation or D. , E. colony number. Quantitation of ES cell colony number shown as mean ± SEM for N = 3.

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Cell Culture, Derivative Assay, Expressing, Flow Cytometry, Cytometry, Incubation, Quantitation Assay

    Cyclin D1 is increased in the stroma of human breast cancer associated with poor outcome A. . The relative abundance of cyclin D1 in either the normal breast ( N = 6) or breast cancer stroma (N = 53) was quantitated as mean ± SEM and shown as either Log2 or B. relative mRNA abundance. C. Kaplan-Meier plot indicating unfavorable prognosis in breast cancer patients with high cyclin D1 in stromal cells. Quantitative immunofluorescence and data-driven dichotomization of nuclear cyclin D1 levels in breast cancer stromal cells is associated with increased risk of disease recurrence ( N = 914, p = 0.03). D. Venn diagram of GO terms, or E. gene expression comparing cancer-associated fibroblast (CAF) genes (breast cancer-associated fibroblasts compared with normal mammary gland fibroblasts [ 34 ] and cyclin D1 induced genes in fibroblasts [ 62 ]). F. Relative number of fibroblasts or G. breast cancer cells determined in co-culture of hTERT fibroblasts (control) or hTERT fibroblasts expressing cyclin D1 (cyclin D1 stroma ) co-incubated with the breast cancer cell line, MDA-MB-231 ( N = 7 for cyclin D1 stroma and N = 12 for control at time 72 hour. N = 2 for all of other time points).

    Journal: Oncotarget

    Article Title: Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth

    doi: 10.18632/oncotarget.19953

    Figure Lengend Snippet: Cyclin D1 is increased in the stroma of human breast cancer associated with poor outcome A. . The relative abundance of cyclin D1 in either the normal breast ( N = 6) or breast cancer stroma (N = 53) was quantitated as mean ± SEM and shown as either Log2 or B. relative mRNA abundance. C. Kaplan-Meier plot indicating unfavorable prognosis in breast cancer patients with high cyclin D1 in stromal cells. Quantitative immunofluorescence and data-driven dichotomization of nuclear cyclin D1 levels in breast cancer stromal cells is associated with increased risk of disease recurrence ( N = 914, p = 0.03). D. Venn diagram of GO terms, or E. gene expression comparing cancer-associated fibroblast (CAF) genes (breast cancer-associated fibroblasts compared with normal mammary gland fibroblasts [ 34 ] and cyclin D1 induced genes in fibroblasts [ 62 ]). F. Relative number of fibroblasts or G. breast cancer cells determined in co-culture of hTERT fibroblasts (control) or hTERT fibroblasts expressing cyclin D1 (cyclin D1 stroma ) co-incubated with the breast cancer cell line, MDA-MB-231 ( N = 7 for cyclin D1 stroma and N = 12 for control at time 72 hour. N = 2 for all of other time points).

    Article Snippet: For immunostaining of cyclin D1 in human breast cancer specimens we have used clinical grade DAKO 3642 rabbit monoclonal antibody specific for the 36 kDa human cyclin D1.

    Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Incubation, Multiple Displacement Amplification

    Proposed Mechanism of PGV-1 Enhanced S-phase Block and DNA Damage by 5-FU. In a sensitive cancer cell, 5-FU is metabolized into active metabolite 5’-dUMP. It interfere the DNA synthesis and causes apoptosis induced by DNA damage (A). In WiDr cell, 5-FU treatment stimulated NF-κB activation then increased cyclin D1 level. High level of cyclin D1 at S-phase leads to repression of DNA synthesis thus it blocks 5-FU effect (B). In the presence of PGV-1, an inhibitor of NF-κB, cyclin D1 level significantly suppressed and leads to the enhancement of S-phase block by 5-FU (C).

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Curcumin Analog Pentagamavunon-1 (PGV-1) Sensitizes Widr Cells to 5-Fluorouracil through Inhibition of NF-κB Activation

    doi: 10.22034/APJCP.2018.19.1.49

    Figure Lengend Snippet: Proposed Mechanism of PGV-1 Enhanced S-phase Block and DNA Damage by 5-FU. In a sensitive cancer cell, 5-FU is metabolized into active metabolite 5’-dUMP. It interfere the DNA synthesis and causes apoptosis induced by DNA damage (A). In WiDr cell, 5-FU treatment stimulated NF-κB activation then increased cyclin D1 level. High level of cyclin D1 at S-phase leads to repression of DNA synthesis thus it blocks 5-FU effect (B). In the presence of PGV-1, an inhibitor of NF-κB, cyclin D1 level significantly suppressed and leads to the enhancement of S-phase block by 5-FU (C).

    Article Snippet: The antibodies used in this study were: anti-α-tubulin, anti-cyclin D1 (Sigma); anti-COX-2 antibody (Dacocytomation, Germany); anti-cyclin A and anti-cyclin B antibodies (BD Biosciences).

    Techniques: Blocking Assay, DNA Synthesis, Activation Assay

    PTL decreased the viability of C918 cells by arresting G1 phase A: PI staining kit was used to measure the percentage of cell number at cell cycle S, G1, and G2 phase in C918 cells treat with different concentration of PTL. B, C: The relative mRNAs and protein expressions of P21 and Cyclin D1 were detected by qRT-PCR (B) and Western blot (C) assays, respectively. GAPDH served as an internal control. Quality one was applied to measure and count the gray value. a P

    Journal: International Journal of Ophthalmology

    Article Title: Parthenolide inhibits the proliferation and induces the apoptosis of human uveal melanoma cells

    doi: 10.18240/ijo.2019.10.03

    Figure Lengend Snippet: PTL decreased the viability of C918 cells by arresting G1 phase A: PI staining kit was used to measure the percentage of cell number at cell cycle S, G1, and G2 phase in C918 cells treat with different concentration of PTL. B, C: The relative mRNAs and protein expressions of P21 and Cyclin D1 were detected by qRT-PCR (B) and Western blot (C) assays, respectively. GAPDH served as an internal control. Quality one was applied to measure and count the gray value. a P

    Article Snippet: The primary antibodies were as follows: anti-P21 (Invitrogen, 33-7000; dilution: 1:800), anti-Cyclin D1 (Invitrogen, 710428; dilution: 1:2000), anti-Bax (Invitrogen, MA5-14006; dilution: 1:1000), anti-Bcl-XL (Invitrogen, MA5-11950; dilution: 1:1500), anti-Bcl-2 (Invitrogen, MA5-11757; dilution: 1:1000), anti-Caspase-3 (Invitrogen, 700182; dilution: 1:700), anti-Caspase-8 (Invitrogen, 710535; dilution: 1:2000), anti-Caspase-9 (Invitrogen, PA5-16358; dilution: 1:1000) and anti-GAPDH (Invitrogen, 39-8600; dilution: 1:1000).

    Techniques: Staining, Concentration Assay, Quantitative RT-PCR, Western Blot