antibodies against p27 Search Results


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  • 92
    Thermo Fisher antibodies against p27
    Mitochondrial <t>p27</t> is sufficient to induce endothelial cell migration. (A, B) Endothelial cells were treated with 50 μM caffeine for 18 hours, and mitochondrial (“mito”) and nonmitochondrial (“non-mito”) fractions were separated. p27 and the closely related p21 protein were detected by immunoblot; TIM23 and Trx-1 served as purity controls for the fractions. (A) Representative immunoblots. (B) Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, n = 6, * p
    Antibodies Against P27, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore anti p27 antibody
    Generation of infectious ALV-J reporter viruses. A, Detection of recombinant genomes in supernatants and cell lysates of rHPRS-103EGFP-infected DF-1 cells by RT-PCR. RT-PCR was conducted using primers that bind to the EGFP ORF. B, Fluorescence microscopy of rALV-J-EGFP-infected cells. Cells were stained with a monoclonal antibody reactive to the ALV-J <t>p27</t> protein, and EGFP expression was examined. Cells were fixed at 48 h postinfection and subjected to indirect immunofluorescence to detect p27 protein (red) and EGFP autofluorescence. The position of the nucleus is indicatedby DAPI (blue) staining in the merged image.
    Anti P27 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson antibodies against p27
    LIF promotes Stat3 phosphorylation to block magnolin-induced autophagy and cell cycle arrest. a HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. The protein levels of p-Stat3 were determined by western blot assays. Total Stat3 expressions were detected as the internal control. b Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec). The levels of LIF, p-Stat3, and Total Stat3 proteins were detected by western blot assays. c Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of p-Stat3 and Total Stat3 proteins were determined by western blot assays. d – j Cells were transfected with Stat3 (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. d , e The protein levels of Stat3 and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. f The protein levels of LC-3B, p62, Cyclin D1, and <t>p27</t> were detected by western blot assays. g , h The cell cycle distribution was determined by flow cytometer. i , j Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. For ( d , e ) and ( g – j ), data are shown as mean ± s.d. ( n = 3); ** P
    Antibodies Against P27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology antibodies against p27
    Modulation of <t>p27</t> and its regulators by Efipladib during cell cycle re-entry A. Quiescent PC-3 cells were induced to re-enter the cell cycle by re-plating them at a low density in 6-well plates. Either 25 μM Efipladib or DMSO was added upon re-plating the cells. Three or five days after treatment, the cells were harvested for immunoblot analysis. No CI: no contact inhibition; 3d CI: contact inhibition for 3 days; 3d D: treatment of re-plated (RP) cells with DMSO (D) for 3 days; 3d E: treatment of re-plated cells with Efipladib (E) for 3 days; 5d D: treatment of re-plated cells with DMSO (D) for 5 days; 5d E: treatment of re-plated cells with Efipladib (E) for 5 days. B. Quiescent LNCaP cells were allowed to re-enter cell cycle by re-plating them in the presence of serum in 6-well plates. Either 20 μM Efipladib or DMSO was administered upon serum restoration. Three or five days after treatment, the cells were collected for immunoblot analysis. No SW: no serum withdrawal; 7d SW: serum withdrawal for 7 days; 3d D: treatment of serum-replenished cells with DMSO (D) for 3 days; 3d E: treatment of serum-replenished cells with Efipladib (E) for 3 days; 5d D: treatment of serum-replenished cells with DMSO (D) for 5 days; 5d E: treatment of serum-replenished cells with Efipladib (E) for 5 days. The results are representative of three experiments.
    Antibodies Against P27, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc antibodies against p27
    ). (c) <t>P27</t> in gastric, lung, and breast cancer (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (d) CDK6 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (e) CCND1 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate.
    Antibodies Against P27, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc p27
    MLN8237 treatment activated the calpain pathway and induced apoptosis through <t>p27</t> degradation and Bax cleavage in AGS cell line. a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f , g MLN8237-induced Ca 2+ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM ( f ). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. ** P
    P27, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 2469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Novocastra monoclonal antibodies against p27
    Representative Sections of Oral SCC Demonstrating Positive Nuclear Immunostaining With Antibodies Against (A) <t>p27</t> and (B) p53 (original magnification × 400) Arrow points to immunostained nuclei of neoplastic cells.
    Monoclonal Antibodies Against P27, supplied by Novocastra, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc monoclonal antibody against p27
    XFC extract inhibits CDK4 and cyclin D3 in SKOV-3 ovarian cancer cells. Human ovarian clear cell carcinoma (ES-2) and human ovarian adenocarcinoma (SKOV-3) cells were cultured as described in Section 2 . Treatment with various concentrations of XFC was performed in serum-free media for 24 hours. Cell lysates were harvested and then processed for (a) SDS-PAGE and western blotting in order to assess the expression levels of Cdk2, CDk4, Cdk6, Cyclin D1, Cyclin D3, <t>p27,</t> and GAPDH. (b) Levels of expression were quantified using scanning densitometry.
    Monoclonal Antibody Against P27, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies monoclonal antibody against p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Monoclonal Antibody Against P27, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam antibody against p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Antibody Against P27, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson mab against p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Mab Against P27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Becton Dickinson antibody against cdkn1b p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Antibody Against Cdkn1b P27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology rabbit anticow polyclonal antibody against p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Rabbit Anticow Polyclonal Antibody Against P27, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Santa Cruz Biotechnology rabbit polyclonal antibody against human p27
    <t>p27</t> expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant
    Rabbit Polyclonal Antibody Against Human P27, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Cell Signaling Technology Inc antibodies against p27 kip1
    Effects of resveratrol and/or TRAIL on Bcl-2 family members and cell cycle regulatory proteins. (A), Western blot analysis was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control, resveratrol and/or TRAIL treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of Bax and Bcl-2 positive cells in tumor cells (right panel). (B), Western blot analysis was performed to measure the expressions of p27 /KIP1  and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of p27 /KIP1  and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of p27 /KIP1  and cyclin D1 positive cells in tumor tissues (right panel).
    Antibodies Against P27 Kip1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson p27
    Transgenic expression of <t>p27</t> driven by the aP2 promoter suppressed cell proliferation in BAT. ( A – C ) Representative images of BAT sections from Tg mice show a lower expression of the cell proliferation markers, PCNA and Ki67, compared to WT mice ( A ). Quantification of PCNA-positive cells ( B ) and western blot analyses ( C ) in the BAT confirmed decreased expression of proliferative markers but there were no essential differences with respect to the apoptosis-related protein (n = 5 per group, Student’s t-test, *p
    P27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p27
    Increased expression of <t>p27</t> in serum-starved Rb1 G/G MEFs. (A) Fibroblast cells of the indicated genotypes were serum starved for 60 h and pulse-labeled with BrdU for 2 h, followed by staining for BrdU incorporation. The proportion of cells incorporating
    P27, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson mouse mab against p27
    Increased expression of <t>p27</t> in serum-starved Rb1 G/G MEFs. (A) Fibroblast cells of the indicated genotypes were serum starved for 60 h and pulse-labeled with BrdU for 2 h, followed by staining for BrdU incorporation. The proportion of cells incorporating
    Mouse Mab Against P27, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Novocastra mouse monoclonal antibody against p27
    Increased expression of <t>p27</t> in serum-starved Rb1 G/G MEFs. (A) Fibroblast cells of the indicated genotypes were serum starved for 60 h and pulse-labeled with BrdU for 2 h, followed by staining for BrdU incorporation. The proportion of cells incorporating
    Mouse Monoclonal Antibody Against P27, supplied by Novocastra, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology p27 kip1
    Knockdown of DICER prolongs the G1/S transition through up-regulating p21 Waf1/Cip1 and <t>p27/</t> <t>Kip1</t> . ( A ) The number of human cells (MRC5SV, HeLa, ATM −/− , M059J and 180BRM) was counted at 72 h after the cells were treated with CtRNA or siDICER. The data were obtained from two independent experiments. ( B ) HeLa cells were collected at 60 h after treatment with CtRNA or siDICER. HeLa cells in the G1 and S phases were detected by combining dye from PI, BrdU labeling and flow cytometry as described in the Supplementary Materials. Similar results were obtained from three independent experiments that the authors carried out. ( C ) Immunoblot detection was performed from MRC5SV and HeLa cells treated with siDICER for 60 h. Similar data were obtained from two independent experiments that the authors carried out. ( D ) HeLa cells were treated with siDICER with or without siRNA against p21 or/and p27 (si-p21, si-p27 or si-p21/si-p27) for 60 h, and the cells were collected for immunoblot detection. Similar data were obtained from two independent experiments. ( E ) The effects of si-p21/si-p27 on the G1-S transition in the DICER knockdowns. HeLa cells were collected for detecting G1 and S phase cell distribution as described in B. Similar results were obtained from three independent experiments that the authors carried out.
    P27 Kip1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc monoclonal rabbit anti human antibody against p27
    Simplified representation of Type I and Type II circuits. The master transcription factors are cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4), and <t>p27</t> is the target gene. Inside each circuit, → indicates transcription activation and ⊥ indicates post-transcriptional repression.
    Monoclonal Rabbit Anti Human Antibody Against P27, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology antibodies against
    Simplified representation of Type I and Type II circuits. The master transcription factors are cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4), and <t>p27</t> is the target gene. Inside each circuit, → indicates transcription activation and ⊥ indicates post-transcriptional repression.
    Antibodies Against, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Mitochondrial p27 is sufficient to induce endothelial cell migration. (A, B) Endothelial cells were treated with 50 μM caffeine for 18 hours, and mitochondrial (“mito”) and nonmitochondrial (“non-mito”) fractions were separated. p27 and the closely related p21 protein were detected by immunoblot; TIM23 and Trx-1 served as purity controls for the fractions. (A) Representative immunoblots. (B) Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, n = 6, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Mitochondrial p27 is sufficient to induce endothelial cell migration. (A, B) Endothelial cells were treated with 50 μM caffeine for 18 hours, and mitochondrial (“mito”) and nonmitochondrial (“non-mito”) fractions were separated. p27 and the closely related p21 protein were detected by immunoblot; TIM23 and Trx-1 served as purity controls for the fractions. (A) Representative immunoblots. (B) Semiquantitative analysis of mitochondrial p27 normalized to TIM23. Data are mean ± SEM, n = 6, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Migration, Western Blot

    The N- and C-terminus of p27 are required for endothelial cell migration and ATP content. (A) Schematic representation of mitochondrially targeted p27 deletion mutants lacking the N-terminus (“ΔN”), the C-terminus (“ΔC”), or both (“ΔNΔC”). The full-length protein (“fl”) and all mutants contain an N-terminal mitochondrial targeting sequence (“MTS,” red) and a C-terminal myc tag (green). Numbers indicate the deletion endpoints within p27. (B-E) Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for the mitochondrially targeted p27 mutants depicted in (A). (B, C) Expression and localization of the mitochondrially targeted mutant p27 proteins were analyzed by immunoblot and immunofluorescence. (B) Representative immunoblot, tubulin served as loading control. (C) Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 mutants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. (D) Migratory capacity was measured in a scratch wound assay by counting cells migrated into the wound using Image J. Data are mean ± SEM, n = 6, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: The N- and C-terminus of p27 are required for endothelial cell migration and ATP content. (A) Schematic representation of mitochondrially targeted p27 deletion mutants lacking the N-terminus (“ΔN”), the C-terminus (“ΔC”), or both (“ΔNΔC”). The full-length protein (“fl”) and all mutants contain an N-terminal mitochondrial targeting sequence (“MTS,” red) and a C-terminal myc tag (green). Numbers indicate the deletion endpoints within p27. (B-E) Endothelial cells were transfected with an empty vector (“EV”) or expression vectors for the mitochondrially targeted p27 mutants depicted in (A). (B, C) Expression and localization of the mitochondrially targeted mutant p27 proteins were analyzed by immunoblot and immunofluorescence. (B) Representative immunoblot, tubulin served as loading control. (C) Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the targeted p27 mutants by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. (D) Migratory capacity was measured in a scratch wound assay by counting cells migrated into the wound using Image J. Data are mean ± SEM, n = 6, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Migration, Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Scratch Wound Assay Assay

    Caffeine effects in the heart depend on p27. (A) The mouse cardiomyocyte cell line HL-1 was lentivirally transduced with an empty vector (“EV”) or an expression vector for mitochondrially targeted p27 (“mito p27”) and treated with 500 μM H 2 O 2 for 48 hours. Apoptosis was measured as annexin V positive/7-PI negative cells by flow cytometry. Data are mean ± SEM, n = 5, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Caffeine effects in the heart depend on p27. (A) The mouse cardiomyocyte cell line HL-1 was lentivirally transduced with an empty vector (“EV”) or an expression vector for mitochondrially targeted p27 (“mito p27”) and treated with 500 μM H 2 O 2 for 48 hours. Apoptosis was measured as annexin V positive/7-PI negative cells by flow cytometry. Data are mean ± SEM, n = 5, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Transduction, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry

    p27 is required for endothelial cell migration. (A, B) p27 was knocked down in endothelial cells by transfection with 2 different siRNAs targeting the p27 mRNA (“p27 siRNA-1,” “p27 siRNA-2”) or a scrambled siRNA (“scr”) as control, and p27 levels were determined by immunoblot. (A) Representative immunoblots, Actin served as loading control. (B) Knockdown efficiency was determined by semiquantitative analysis of immunoblots. Data are mean ± SEM, n = 5, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: p27 is required for endothelial cell migration. (A, B) p27 was knocked down in endothelial cells by transfection with 2 different siRNAs targeting the p27 mRNA (“p27 siRNA-1,” “p27 siRNA-2”) or a scrambled siRNA (“scr”) as control, and p27 levels were determined by immunoblot. (A) Representative immunoblots, Actin served as loading control. (B) Knockdown efficiency was determined by semiquantitative analysis of immunoblots. Data are mean ± SEM, n = 5, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Migration, Transfection, Western Blot

    Serine 10 and threonine 187 of p27 are required for endothelial cell migration and mitochondrial import. (A, B) Endothelial cells were treated with caffeine for 18 hours or left untreated, and phosphorylation of serine 10 (“p27 P-S10”) and threonine 187 (“p27 P-T187), as well as total p27 (“p27”), were detected by immunoblot. (A) Representative immunoblots with the corresponding loading control (Tubulin) below the respective immunoblot. The asterisk denotes p27 phosphorylated on threonine 187. (B) Semiquantitative analyses of the ratio of phosphorylated p27 to total p27 for both phosphorylation events. Data are mean ± SEM, n = 7: p27 P-S10, n = 6: p27 P-T187, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Serine 10 and threonine 187 of p27 are required for endothelial cell migration and mitochondrial import. (A, B) Endothelial cells were treated with caffeine for 18 hours or left untreated, and phosphorylation of serine 10 (“p27 P-S10”) and threonine 187 (“p27 P-T187), as well as total p27 (“p27”), were detected by immunoblot. (A) Representative immunoblots with the corresponding loading control (Tubulin) below the respective immunoblot. The asterisk denotes p27 phosphorylated on threonine 187. (B) Semiquantitative analyses of the ratio of phosphorylated p27 to total p27 for both phosphorylation events. Data are mean ± SEM, n = 7: p27 P-S10, n = 6: p27 P-T187, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Migration, Western Blot

    Mitochondrial p27 restores the impaired αSMA up-regulation in p27-deficient cardiac fibroblasts. Fibroblasts isolated from the hearts of p27-deficient mice were lentivirally transduced with an expression vector for mitochondrially targeted p27 (“mito p27”) or a corresponding empty vector (“EV”). (A) Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the mitochondrially targeted p27 by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. (B, C) Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours, and αSMA was detected by immunoblot. (B) Representative immunoblots, Vimentin served as loading control. (C) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 5, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Mitochondrial p27 restores the impaired αSMA up-regulation in p27-deficient cardiac fibroblasts. Fibroblasts isolated from the hearts of p27-deficient mice were lentivirally transduced with an expression vector for mitochondrially targeted p27 (“mito p27”) or a corresponding empty vector (“EV”). (A) Representative immunostainings: nuclei were visualized with DAPI (blue), mitochondria by staining for TIM23 (red), and the mitochondrially targeted p27 by staining for the myc epitope (“myc (p27),” green). Merge shows an overlay of all fluorescence channels. (B, C) Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours, and αSMA was detected by immunoblot. (B) Representative immunoblots, Vimentin served as loading control. (C) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 5, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Isolation, Mouse Assay, Transduction, Expressing, Plasmid Preparation, Staining, Fluorescence, Western Blot

    p27 is required for myofibroblast differentiation of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (“wt”) mice and p27-deficient (“p27 −/−”) littermates. Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours in the presence or absence of 50 μM caffeine. Induction of αSMA was detected by immunoblot and immunostaining. (A) Representative immunoblots, Vimentin served as loading control. (B) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 8: wt untreated, wt +TGFβ1, p27 −/− untreated, p27 −/− +TGFβ1; n = 5: all others, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: p27 is required for myofibroblast differentiation of cardiac fibroblasts. Cardiac fibroblasts were isolated from hearts of wild-type (“wt”) mice and p27-deficient (“p27 −/−”) littermates. Myofibroblast differentiation was induced by treatment with 2 ng/ml TGFβ1 for 48 hours in the presence or absence of 50 μM caffeine. Induction of αSMA was detected by immunoblot and immunostaining. (A) Representative immunoblots, Vimentin served as loading control. (B) Semiquantitative analysis of αSMA normalized to Vimentin. Data are mean ± SEM, n = 8: wt untreated, wt +TGFβ1, p27 −/− untreated, p27 −/− +TGFβ1; n = 5: all others, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Isolation, Mouse Assay, Immunostaining, Western Blot

    Caffeine enhances respiration, ATP content, and mitochondrial localization of p27 in old mouse hearts. (A-D) Twenty-two-month-old wild-type mice received drinking water (“old”) or water supplemented with 0.05% caffeine for 10 days (“old+caffeine”). (A) O 2 consumption was measured in isolated heart mitochondria without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O 2 consumption after the addition of malate/glutamate and ADP, respectively. Data are mean ± SEM, n = 6 per group, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Caffeine enhances respiration, ATP content, and mitochondrial localization of p27 in old mouse hearts. (A-D) Twenty-two-month-old wild-type mice received drinking water (“old”) or water supplemented with 0.05% caffeine for 10 days (“old+caffeine”). (A) O 2 consumption was measured in isolated heart mitochondria without the addition of substrates (“mito”) and after the successive addition of malate/glutamate (“M/G”), ADP, rotenone (“rot”), and succinate (“succ”) (left panel). The right panel shows a magnification of O 2 consumption after the addition of malate/glutamate and ADP, respectively. Data are mean ± SEM, n = 6 per group, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Mouse Assay, Isolation

    Caffeine improves outcomes after myocardial infarction in prediabetic mice and induces mitochondrial translocation of p27. Two-month-old wild-type mice were fed a diabetogenic diet for 11 weeks. For the last 10 days, one group of animals received drinking water supplemented with 0.05% caffeine. Afterward, myocardial infarction was induced by ligation of the left anterior descending coronary artery for 60 minutes followed by reperfusion. Twenty-one days after infarction, hearts were excised, sectioned, and the sections stained. (A) Representative Gomori stainings of sections of 3 different hearts for each dietary regimen. (B) Infarct size per left ventricle and (C) minimum left ventricular (“LV”) wall thickness in the infarcted myocardium. Data are mean ± SEM, n = 8: diabetogenic diet, n = 10: diabetogenic diet +caffeine, * p

    Journal: PLoS Biology

    Article Title: CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system—New mode of action for caffeine

    doi: 10.1371/journal.pbio.2004408

    Figure Lengend Snippet: Caffeine improves outcomes after myocardial infarction in prediabetic mice and induces mitochondrial translocation of p27. Two-month-old wild-type mice were fed a diabetogenic diet for 11 weeks. For the last 10 days, one group of animals received drinking water supplemented with 0.05% caffeine. Afterward, myocardial infarction was induced by ligation of the left anterior descending coronary artery for 60 minutes followed by reperfusion. Twenty-one days after infarction, hearts were excised, sectioned, and the sections stained. (A) Representative Gomori stainings of sections of 3 different hearts for each dietary regimen. (B) Infarct size per left ventricle and (C) minimum left ventricular (“LV”) wall thickness in the infarcted myocardium. Data are mean ± SEM, n = 8: diabetogenic diet, n = 10: diabetogenic diet +caffeine, * p

    Article Snippet: Immunostaining of heart slices The sections were stained with antibodies against p27 (polyclonal, PA5-27188, Thermo Fisher Scientific, 1:25) and TIM23 (clone 32, BD Biosciences, 1:100).

    Techniques: Mouse Assay, Translocation Assay, Ligation, Staining

    Generation of infectious ALV-J reporter viruses. A, Detection of recombinant genomes in supernatants and cell lysates of rHPRS-103EGFP-infected DF-1 cells by RT-PCR. RT-PCR was conducted using primers that bind to the EGFP ORF. B, Fluorescence microscopy of rALV-J-EGFP-infected cells. Cells were stained with a monoclonal antibody reactive to the ALV-J p27 protein, and EGFP expression was examined. Cells were fixed at 48 h postinfection and subjected to indirect immunofluorescence to detect p27 protein (red) and EGFP autofluorescence. The position of the nucleus is indicatedby DAPI (blue) staining in the merged image.

    Journal: PLoS ONE

    Article Title: A Recombinant Avian Leukosis Virus Subgroup J for Directly Monitoring Viral Infection and the Selection of Neutralizing Antibodies

    doi: 10.1371/journal.pone.0115422

    Figure Lengend Snippet: Generation of infectious ALV-J reporter viruses. A, Detection of recombinant genomes in supernatants and cell lysates of rHPRS-103EGFP-infected DF-1 cells by RT-PCR. RT-PCR was conducted using primers that bind to the EGFP ORF. B, Fluorescence microscopy of rALV-J-EGFP-infected cells. Cells were stained with a monoclonal antibody reactive to the ALV-J p27 protein, and EGFP expression was examined. Cells were fixed at 48 h postinfection and subjected to indirect immunofluorescence to detect p27 protein (red) and EGFP autofluorescence. The position of the nucleus is indicatedby DAPI (blue) staining in the merged image.

    Article Snippet: The cells were then incubated with anti-p27 antibody for 2h followed by incubation with goat anti-mouse IgG (whole molecule)–TRITC antibody (Sigma, USA).

    Techniques: Recombinant, Infection, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Staining, Expressing, Immunofluorescence

    Replication of rHPRS-103EGFP and rHPRS-103. A, Growth kinetics assay of rHPRS-103EGFP and rHPRS-103. B, The growth kinetics of rHPRS-103EGFP was measured by p27 expression using IFA while the fluorescence is a direct measure of EGFP-positive cells.

    Journal: PLoS ONE

    Article Title: A Recombinant Avian Leukosis Virus Subgroup J for Directly Monitoring Viral Infection and the Selection of Neutralizing Antibodies

    doi: 10.1371/journal.pone.0115422

    Figure Lengend Snippet: Replication of rHPRS-103EGFP and rHPRS-103. A, Growth kinetics assay of rHPRS-103EGFP and rHPRS-103. B, The growth kinetics of rHPRS-103EGFP was measured by p27 expression using IFA while the fluorescence is a direct measure of EGFP-positive cells.

    Article Snippet: The cells were then incubated with anti-p27 antibody for 2h followed by incubation with goat anti-mouse IgG (whole molecule)–TRITC antibody (Sigma, USA).

    Techniques: Expressing, Immunofluorescence, Fluorescence

    LIF promotes Stat3 phosphorylation to block magnolin-induced autophagy and cell cycle arrest. a HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. The protein levels of p-Stat3 were determined by western blot assays. Total Stat3 expressions were detected as the internal control. b Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec). The levels of LIF, p-Stat3, and Total Stat3 proteins were detected by western blot assays. c Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of p-Stat3 and Total Stat3 proteins were determined by western blot assays. d – j Cells were transfected with Stat3 (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. d , e The protein levels of Stat3 and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. f The protein levels of LC-3B, p62, Cyclin D1, and p27 were detected by western blot assays. g , h The cell cycle distribution was determined by flow cytometer. i , j Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. For ( d , e ) and ( g – j ), data are shown as mean ± s.d. ( n = 3); ** P

    Journal: Cell Death & Disease

    Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

    doi: 10.1038/s41419-018-0660-4

    Figure Lengend Snippet: LIF promotes Stat3 phosphorylation to block magnolin-induced autophagy and cell cycle arrest. a HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. The protein levels of p-Stat3 were determined by western blot assays. Total Stat3 expressions were detected as the internal control. b Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec). The levels of LIF, p-Stat3, and Total Stat3 proteins were detected by western blot assays. c Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of p-Stat3 and Total Stat3 proteins were determined by western blot assays. d – j Cells were transfected with Stat3 (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. d , e The protein levels of Stat3 and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. f The protein levels of LC-3B, p62, Cyclin D1, and p27 were detected by western blot assays. g , h The cell cycle distribution was determined by flow cytometer. i , j Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. For ( d , e ) and ( g – j ), data are shown as mean ± s.d. ( n = 3); ** P

    Article Snippet: Antibodies against p27 and Cyclin D1 were purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: Blocking Assay, Western Blot, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Laser-Scanning Microscopy

    Magnolin inhibits growth and induces cell cycle arrest in CRC cells. a Chemical structure of magnolin. b Cell viability was examined using MTT assay in HCT116 and SW480 cells treated with magnolin at different concentrations for 48 h. c The clonogenicity of HCT116 and SW480 cells were detected after treatment with magnolin at different concentrations for 14 days. d , e HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. Cell cycle distribution was determined by flow cytometer. f The protein levels of Cyclin D1, p27, and Cyclin B1 were determined by western blot assays. g Cyclin D1 and p27 levels in xenograft tumors were examined by western blot assays. h Ki67 and Cyclin D1 expressions in xenograft tumors were examined by IHC staining. Representative images were conducted as indicated. *** P

    Journal: Cell Death & Disease

    Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

    doi: 10.1038/s41419-018-0660-4

    Figure Lengend Snippet: Magnolin inhibits growth and induces cell cycle arrest in CRC cells. a Chemical structure of magnolin. b Cell viability was examined using MTT assay in HCT116 and SW480 cells treated with magnolin at different concentrations for 48 h. c The clonogenicity of HCT116 and SW480 cells were detected after treatment with magnolin at different concentrations for 14 days. d , e HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. Cell cycle distribution was determined by flow cytometer. f The protein levels of Cyclin D1, p27, and Cyclin B1 were determined by western blot assays. g Cyclin D1 and p27 levels in xenograft tumors were examined by western blot assays. h Ki67 and Cyclin D1 expressions in xenograft tumors were examined by IHC staining. Representative images were conducted as indicated. *** P

    Article Snippet: Antibodies against p27 and Cyclin D1 were purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: MTT Assay, Flow Cytometry, Cytometry, Western Blot, Immunohistochemistry, Staining

    Magnolin inhibits Mcl-1 through inactivation of the LIF signaling. a, b HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. a The protein levels of Mcl-1 were determined by western blot assays. b The mRNA levels of Mcl-1 were detected by real-time PCR. c Cells were transfected with Mcl-1 (Mcl-1 Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of Mcl-1, LC-3B, p62, Cyclin D1, and p27 proteins were detected by western blot assays. d The protein and mRNA levels of LIF were detected by western blot assays and real-time PCR. e – i Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. e , f The protein levels of LIF and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. g , h Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. Scale bar, 20 μm. i The cell cycle distribution was determined by flow cytometer. j , k Cells were transfected with control siRNA or siRNA against LIF. j The levels of LIF and Mcl-1 proteins were determined by western blot assays. k The levels of LC-3B, p62, Cyclin D1, and p27 proteins were detected by western blot assays. For ( b ) and ( d ), data are shown as mean ± s.d. ( n = 3); * P

    Journal: Cell Death & Disease

    Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

    doi: 10.1038/s41419-018-0660-4

    Figure Lengend Snippet: Magnolin inhibits Mcl-1 through inactivation of the LIF signaling. a, b HCT116 and SW480 cells were treated with indicated concentrations of magnolin for 48 h. a The protein levels of Mcl-1 were determined by western blot assays. b The mRNA levels of Mcl-1 were detected by real-time PCR. c Cells were transfected with Mcl-1 (Mcl-1 Vec) or empty vector (Control Vec) and followed by magnolin treatment. The levels of Mcl-1, LC-3B, p62, Cyclin D1, and p27 proteins were detected by western blot assays. d The protein and mRNA levels of LIF were detected by western blot assays and real-time PCR. e – i Cells were transfected with LIF (LIF Vec) or empty vector (Control Vec) and followed by magnolin treatment. e , f The protein levels of LIF and Mcl-1 were determined by western blot assays. The mRNA levels of Mcl-1 were detected by real-time PCR. g , h Cells were transfected with a reporter plasmid (mRFP-GFP-LC3), followed by a confocal laser scanning microscope. Scale bar, 20 μm. i The cell cycle distribution was determined by flow cytometer. j , k Cells were transfected with control siRNA or siRNA against LIF. j The levels of LIF and Mcl-1 proteins were determined by western blot assays. k The levels of LC-3B, p62, Cyclin D1, and p27 proteins were detected by western blot assays. For ( b ) and ( d ), data are shown as mean ± s.d. ( n = 3); * P

    Article Snippet: Antibodies against p27 and Cyclin D1 were purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, Laser-Scanning Microscopy, Flow Cytometry, Cytometry

    Inhibition of autophagy blocks magnolin-induced cell cycle arrest. a , b Cells were treated with magnolin with or without 3-MA respectively. a The levels of LC-3B, p62, Cyclin D1, and p27 proteins were examined by western blot assays. b The cell cycle distribution was determined by flow cytometer. c , d Cells were transfected with control siRNA or siRNA against LC-3B followed by magnolin treatment. c The levels of LC-3B, p62, Cyclin D1, and p27 proteins were examined by western blot assays. d The cell cycle distribution was determined by flow cytometer. For ( b ) and ( d ), data are shown as mean ± s.d. ( n = 3); ** P

    Journal: Cell Death & Disease

    Article Title: Magnolin promotes autophagy and cell cycle arrest via blocking LIF/Stat3/Mcl-1 axis in human colorectal cancers

    doi: 10.1038/s41419-018-0660-4

    Figure Lengend Snippet: Inhibition of autophagy blocks magnolin-induced cell cycle arrest. a , b Cells were treated with magnolin with or without 3-MA respectively. a The levels of LC-3B, p62, Cyclin D1, and p27 proteins were examined by western blot assays. b The cell cycle distribution was determined by flow cytometer. c , d Cells were transfected with control siRNA or siRNA against LC-3B followed by magnolin treatment. c The levels of LC-3B, p62, Cyclin D1, and p27 proteins were examined by western blot assays. d The cell cycle distribution was determined by flow cytometer. For ( b ) and ( d ), data are shown as mean ± s.d. ( n = 3); ** P

    Article Snippet: Antibodies against p27 and Cyclin D1 were purchased from BD Biosciences (San Jose, CA, USA).

    Techniques: Inhibition, Western Blot, Flow Cytometry, Cytometry, Transfection

    Modulation of p27 and its regulators by Efipladib during cell cycle re-entry A. Quiescent PC-3 cells were induced to re-enter the cell cycle by re-plating them at a low density in 6-well plates. Either 25 μM Efipladib or DMSO was added upon re-plating the cells. Three or five days after treatment, the cells were harvested for immunoblot analysis. No CI: no contact inhibition; 3d CI: contact inhibition for 3 days; 3d D: treatment of re-plated (RP) cells with DMSO (D) for 3 days; 3d E: treatment of re-plated cells with Efipladib (E) for 3 days; 5d D: treatment of re-plated cells with DMSO (D) for 5 days; 5d E: treatment of re-plated cells with Efipladib (E) for 5 days. B. Quiescent LNCaP cells were allowed to re-enter cell cycle by re-plating them in the presence of serum in 6-well plates. Either 20 μM Efipladib or DMSO was administered upon serum restoration. Three or five days after treatment, the cells were collected for immunoblot analysis. No SW: no serum withdrawal; 7d SW: serum withdrawal for 7 days; 3d D: treatment of serum-replenished cells with DMSO (D) for 3 days; 3d E: treatment of serum-replenished cells with Efipladib (E) for 3 days; 5d D: treatment of serum-replenished cells with DMSO (D) for 5 days; 5d E: treatment of serum-replenished cells with Efipladib (E) for 5 days. The results are representative of three experiments.

    Journal: Oncotarget

    Article Title: Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells

    doi:

    Figure Lengend Snippet: Modulation of p27 and its regulators by Efipladib during cell cycle re-entry A. Quiescent PC-3 cells were induced to re-enter the cell cycle by re-plating them at a low density in 6-well plates. Either 25 μM Efipladib or DMSO was added upon re-plating the cells. Three or five days after treatment, the cells were harvested for immunoblot analysis. No CI: no contact inhibition; 3d CI: contact inhibition for 3 days; 3d D: treatment of re-plated (RP) cells with DMSO (D) for 3 days; 3d E: treatment of re-plated cells with Efipladib (E) for 3 days; 5d D: treatment of re-plated cells with DMSO (D) for 5 days; 5d E: treatment of re-plated cells with Efipladib (E) for 5 days. B. Quiescent LNCaP cells were allowed to re-enter cell cycle by re-plating them in the presence of serum in 6-well plates. Either 20 μM Efipladib or DMSO was administered upon serum restoration. Three or five days after treatment, the cells were collected for immunoblot analysis. No SW: no serum withdrawal; 7d SW: serum withdrawal for 7 days; 3d D: treatment of serum-replenished cells with DMSO (D) for 3 days; 3d E: treatment of serum-replenished cells with Efipladib (E) for 3 days; 5d D: treatment of serum-replenished cells with DMSO (D) for 5 days; 5d E: treatment of serum-replenished cells with Efipladib (E) for 5 days. The results are representative of three experiments.

    Article Snippet: The primary antibodies against p27 (sc-528), CRM1 (sc-74454), Pirh2 (sc-67033), Skp2 (sc-7164), Cyclin E (sc-198), CDK2 (sc-748), CDK4 (sc-749), CDC6 (sc-9964), MCM7 (sc-22782), ORC6 (sc-20636), PCNA (sc-56), cPLA2 α (sc-454) and phospho-cPLA2 at Ser505 (sc-34391-R) were purchased from Santa Cruz Biotechnology.

    Techniques: Inhibition

    Distribution of p27- and PCAF-BSs on the chromatin. ( A ) Distribution of different chromatin regions overall genome of HCT116 cells (upper panel), p27-BSs (Bottom panel, left) and PCAF-BSs (Bottom panel, right) obtained by ChIP-seq experiments. Results are represented as percentage. ( B ) The different biotypes of genes putatively regulated by p27 (left) or PCAF (right) are represented as percentage.

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: Distribution of p27- and PCAF-BSs on the chromatin. ( A ) Distribution of different chromatin regions overall genome of HCT116 cells (upper panel), p27-BSs (Bottom panel, left) and PCAF-BSs (Bottom panel, right) obtained by ChIP-seq experiments. Results are represented as percentage. ( B ) The different biotypes of genes putatively regulated by p27 (left) or PCAF (right) are represented as percentage.

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Chromatin Immunoprecipitation

    Effect of knocking down p27 or PCAF on the expression of target genes. ( A ) HCT116 cells were infected with a specific shRNA for p27 (shp27) or with a control shRNA (sh(–)). Then, mRNA levels of different common target genes (see Table 1 ) were determined by qPCR. In these cells, the levels of primary non spliced transcripts (PNST) were also determined by qPCR ( B ). ( C ) HCT116 cells were infected with a specific shRNA for PCAF (shPCAF) or with a control shRNA (sh(–)) and the levels of mRNA of different common target genes were subsequently determined by qPCR. In these cells, the levels of primary non spliced transcripts (PNST) were also determined by qPCR ( D ). Levels of mRNA ( E ) or of primary non spliced transcripts (PNST) ( F ) from different common target genes were determined in p27 knocked down cells using the CRISPR/Cas9 (CR p27) methodology and in control cells (CR Ctrl). Levels of mRNA ( G ) or of primary non spliced transcripts (PNST) ( H ) from different common target genes were determined in PACF knocked down cells using the CRISPR/Cas9 (CR PCAF) methodology and in control cells (CR Ctrl). In all experiments results are the mean value ± SEM of four independent experiments and are represented as fold enrichment versus control. Statistical analyses were performed using the t -student's test. * P

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: Effect of knocking down p27 or PCAF on the expression of target genes. ( A ) HCT116 cells were infected with a specific shRNA for p27 (shp27) or with a control shRNA (sh(–)). Then, mRNA levels of different common target genes (see Table 1 ) were determined by qPCR. In these cells, the levels of primary non spliced transcripts (PNST) were also determined by qPCR ( B ). ( C ) HCT116 cells were infected with a specific shRNA for PCAF (shPCAF) or with a control shRNA (sh(–)) and the levels of mRNA of different common target genes were subsequently determined by qPCR. In these cells, the levels of primary non spliced transcripts (PNST) were also determined by qPCR ( D ). Levels of mRNA ( E ) or of primary non spliced transcripts (PNST) ( F ) from different common target genes were determined in p27 knocked down cells using the CRISPR/Cas9 (CR p27) methodology and in control cells (CR Ctrl). Levels of mRNA ( G ) or of primary non spliced transcripts (PNST) ( H ) from different common target genes were determined in PACF knocked down cells using the CRISPR/Cas9 (CR PCAF) methodology and in control cells (CR Ctrl). In all experiments results are the mean value ± SEM of four independent experiments and are represented as fold enrichment versus control. Statistical analyses were performed using the t -student's test. * P

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Expressing, Infection, shRNA, Real-time Polymerase Chain Reaction, CRISPR

    Effect of knocking down both, p27 and PCAF on the expression of target genes. ( A ) HCT116 cells were infected with a specific shRNA for p27 alone, or with shRNAs for p27 and PCAF or with a control shRNA (sh(–)). Then, the levels of primary non spliced transcripts (PNST) were determined by qPCR. ( B ). Cells were Knocked down for p27 and PCAF (double knockout) (2KD) using the CRISPR/Cas9 methodology. Then, the levels of PCAF and p27 were determined by WB. Levels of mRNA ( C ) or of primary non spliced transcripts (PNST) ( D ) from different common target genes were determined in double knock down cells (CR 2KD) and in control cells (CR Ctrl) by qPCR. In all experiments results are the mean value ± SEM of four independent experiments and are represented as fold enrichment versus control. Statistical analyses were performed using the t -student's test. * P

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: Effect of knocking down both, p27 and PCAF on the expression of target genes. ( A ) HCT116 cells were infected with a specific shRNA for p27 alone, or with shRNAs for p27 and PCAF or with a control shRNA (sh(–)). Then, the levels of primary non spliced transcripts (PNST) were determined by qPCR. ( B ). Cells were Knocked down for p27 and PCAF (double knockout) (2KD) using the CRISPR/Cas9 methodology. Then, the levels of PCAF and p27 were determined by WB. Levels of mRNA ( C ) or of primary non spliced transcripts (PNST) ( D ) from different common target genes were determined in double knock down cells (CR 2KD) and in control cells (CR Ctrl) by qPCR. In all experiments results are the mean value ± SEM of four independent experiments and are represented as fold enrichment versus control. Statistical analyses were performed using the t -student's test. * P

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Expressing, Infection, shRNA, Real-time Polymerase Chain Reaction, Double Knockout, CRISPR, Western Blot

    Identification of genes putatively regulated by p27 and PCAF. ( A ) Venn diagram representing the number of genes encoding for proteins identified by ChIP-seq with anti-PCAF antibodies (pink) or with anti-p27 (blue). ( B ) The biological processes enriched in the group of genes sharing p27 and PCAF-BSs (269 genes) were identified by using the DAVID program. ( C ) Number of genes differentially expressed in PCAF-KD versus control HCT116 cells identified by expression microarray analysis and main characteristics of the data. ( D ) The biological processes enriched in the group of genes differentially expressed in PCAF-KD cells versus control were identified using the DAVID program. ( E ) Venn diagram representing the number of genes encoding for proteins identified by ChIP-seq with anti-PCAF antibodies (pink), with anti-p27 (blue) or differentially expressed in PCAF-KD cells (green).

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: Identification of genes putatively regulated by p27 and PCAF. ( A ) Venn diagram representing the number of genes encoding for proteins identified by ChIP-seq with anti-PCAF antibodies (pink) or with anti-p27 (blue). ( B ) The biological processes enriched in the group of genes sharing p27 and PCAF-BSs (269 genes) were identified by using the DAVID program. ( C ) Number of genes differentially expressed in PCAF-KD versus control HCT116 cells identified by expression microarray analysis and main characteristics of the data. ( D ) The biological processes enriched in the group of genes differentially expressed in PCAF-KD cells versus control were identified using the DAVID program. ( E ) Venn diagram representing the number of genes encoding for proteins identified by ChIP-seq with anti-PCAF antibodies (pink), with anti-p27 (blue) or differentially expressed in PCAF-KD cells (green).

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Chromatin Immunoprecipitation, Expressing, Microarray

    Gene ontology analysis of protein encoding genes with p27- or PCAF-BSs in their proximity. The Database for Annotation, Visualization and Integrated Discovery (DAVID) program was used to define biological processes enriched in the protein encoding genes putatively regulated by p27 ( A ), PCAF (intragenic BSs) ( B ) and PCAF (intergenic BSs) ( C ). ( D ) The Gitools program was used to define biological processes enriched in the protein encoding genes putatively regulated by p27 or PCAF. ( E ) MEME programs were used to identify short sequence motifs enriched in the p27-BSs (upper panel) or PCAF-BSs (bottom panel).

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: Gene ontology analysis of protein encoding genes with p27- or PCAF-BSs in their proximity. The Database for Annotation, Visualization and Integrated Discovery (DAVID) program was used to define biological processes enriched in the protein encoding genes putatively regulated by p27 ( A ), PCAF (intragenic BSs) ( B ) and PCAF (intergenic BSs) ( C ). ( D ) The Gitools program was used to define biological processes enriched in the protein encoding genes putatively regulated by p27 or PCAF. ( E ) MEME programs were used to identify short sequence motifs enriched in the p27-BSs (upper panel) or PCAF-BSs (bottom panel).

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Sequencing

    PAX5 collaborates in the regulation of p27- and PCAF-target genes. ( A ) Enrichment analysis of the putative transcription factors that could significantly associate to p27- or PCAF-BSs was performed using GiTools, the STORM tool for the scanning of the peaks and the TF-BSs motif position weight matrix from TRANSFAC. ( B ) IP experiments were performed using anti-PAX5 antibodies or IgG as a control. The presence of p27, PAX5 and PCAF in the inputs and in the immunoprecipitates was determined by WB. ( C ) The interactions of p27 (upper panel) or PCAF (bottom panel) with their specific chromatin binding sites in several target genes were performed by ChIP using anti-p27, anti-PCAF or no-antibodies as a control. Results are represented relative to the control. ( D ) The interaction of PAX5 with the p27- BSs (upper panel) and PCAF-BSs (bottom panel) of the target genes was analyzed by ChIP using anti-PAX5 antibodies or no-antibodies as a control. Results are represented relative to the control ( E ) Cells were infected with a specific shRNA for PAX5 or a control shRNA. Then, the levels of PAX5 mRNA (upper panel) and PAX5 protein (bottom panel) were determined by qPCR or WB, respectively. ( F ) The mRNA levels of different target genes were determined by qPCR in cells infected with a specific shRNA for PAX5 (white bars) or a control shRNA (black bars). Results are represented as a fold change relative to the control. In all cases, results are the mean value ± SD of three independent experiments. Statistical analyses were performed using the t -student's test. * P

    Journal: Nucleic Acids Research

    Article Title: p27Kip1, PCAF and PAX5 cooperate in the transcriptional regulation of specific target genes

    doi: 10.1093/nar/gkx075

    Figure Lengend Snippet: PAX5 collaborates in the regulation of p27- and PCAF-target genes. ( A ) Enrichment analysis of the putative transcription factors that could significantly associate to p27- or PCAF-BSs was performed using GiTools, the STORM tool for the scanning of the peaks and the TF-BSs motif position weight matrix from TRANSFAC. ( B ) IP experiments were performed using anti-PAX5 antibodies or IgG as a control. The presence of p27, PAX5 and PCAF in the inputs and in the immunoprecipitates was determined by WB. ( C ) The interactions of p27 (upper panel) or PCAF (bottom panel) with their specific chromatin binding sites in several target genes were performed by ChIP using anti-p27, anti-PCAF or no-antibodies as a control. Results are represented relative to the control. ( D ) The interaction of PAX5 with the p27- BSs (upper panel) and PCAF-BSs (bottom panel) of the target genes was analyzed by ChIP using anti-PAX5 antibodies or no-antibodies as a control. Results are represented relative to the control ( E ) Cells were infected with a specific shRNA for PAX5 or a control shRNA. Then, the levels of PAX5 mRNA (upper panel) and PAX5 protein (bottom panel) were determined by qPCR or WB, respectively. ( F ) The mRNA levels of different target genes were determined by qPCR in cells infected with a specific shRNA for PAX5 (white bars) or a control shRNA (black bars). Results are represented as a fold change relative to the control. In all cases, results are the mean value ± SD of three independent experiments. Statistical analyses were performed using the t -student's test. * P

    Article Snippet: Antibodies Antibodies against p27 (ChIP and IP) (sc-528), PAX-5 (WB) (sc-13146), PAX-5 (WB) (sc-1974), UNC5D (WB) (anti-UNC5H4, sc-135262), PCAF (WB) (sc-13124), SPAG 9 (WB) (anti- JIP-4, sc-134972) and ROBO1 (WB) (sc-25672) were from Santa Cruz. p27 antibody (WB) (610242) was from BD Transduction Laboratories.

    Techniques: Western Blot, Binding Assay, Chromatin Immunoprecipitation, Infection, shRNA, Real-time Polymerase Chain Reaction

    ). (c) P27 in gastric, lung, and breast cancer (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (d) CDK6 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (e) CCND1 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: ). (c) P27 in gastric, lung, and breast cancer (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (d) CDK6 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate. (e) CCND1 in gastric, lung, and breast cancers (Kaplan–Meier Plotter). Kaplan–Meier plots showing OS in adenocarcinomas. In red: patients with expression above the median and in black, patients with expressions below the median. Kaplan–Meier plots revealed the correlation between gene expression and OS rate.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Expressing

    P27, CDK6 and CCND1 regulate the cell cycle in A549 cells. (a) Flow detection of cell cycle after changes to p27 expression. (b) Flow detection of cell cycle after changes to CDK6 expression. (c) Flow detection of cell cycle after changes to CCND1 expression. (d) Flow detection of cell cycle after changes to p27, CDK6, CCND1 expression

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: P27, CDK6 and CCND1 regulate the cell cycle in A549 cells. (a) Flow detection of cell cycle after changes to p27 expression. (b) Flow detection of cell cycle after changes to CDK6 expression. (c) Flow detection of cell cycle after changes to CCND1 expression. (d) Flow detection of cell cycle after changes to p27, CDK6, CCND1 expression

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Flow Cytometry, Expressing

    The roles of p27 in cell cycle regulation and tumor development. (a) The mechanism of p27 in cell cycle. P27 is ubiquitylated and degraded in late G1, S and G2 phases by SCFSkp2 in the nucleus. P27 phosphorylated at S10 is ubiquitylated by the KPC complex when exported to the cytoplasm. (b) Contributions of the study of cell cycle regulation by P27. The mechanism of p27 in cell cycle. P27 induces cell cycle arrest in the G1 phase by inhibiting the formation of cell cycle-dependent complexes CDK6/CCND1.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: The roles of p27 in cell cycle regulation and tumor development. (a) The mechanism of p27 in cell cycle. P27 is ubiquitylated and degraded in late G1, S and G2 phases by SCFSkp2 in the nucleus. P27 phosphorylated at S10 is ubiquitylated by the KPC complex when exported to the cytoplasm. (b) Contributions of the study of cell cycle regulation by P27. The mechanism of p27 in cell cycle. P27 induces cell cycle arrest in the G1 phase by inhibiting the formation of cell cycle-dependent complexes CDK6/CCND1.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques:

    P27, CDK6 and CCND1 affected cell proliferation in A549 cells. (a) qPCR was used to detect the expression of p27, CDK6 and CCND1 after changing the expression levels of these genes. (b) The number of cells was detected by flow cytometry at 0, 24, 36, and 48 h after changing the expression of the related genes. Control: blank load transfection group as control group; P27-oe: over-expressed P27 in A549; P27-RNAi: Short Hairpin RNA on Expression of P27 in A549; CDK6-oe: over-expressed CDK6 in A549; CDK6-RNAi: Short Hairpin RNA on Expression of CDK6 in A549; CCND1-oe: over-expressed CCND1 in A549; CCND1-RNAi: Short Hairpin RNA on Expression of CCND1 in A549. Horizontal axis represents the hours after cell transfection. The vertical axis represents the cell number. (c) MTT reduction assay was used to reflect the proliferation at 0, 24, 36, and 48 h after changing the expression of related genes. Horizontal axis represents the hours after cell transfection. The vertical axis represents the OD value of cell proliferation. (d) Flow cytometry and MTT methods were used to detect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: P27, CDK6 and CCND1 affected cell proliferation in A549 cells. (a) qPCR was used to detect the expression of p27, CDK6 and CCND1 after changing the expression levels of these genes. (b) The number of cells was detected by flow cytometry at 0, 24, 36, and 48 h after changing the expression of the related genes. Control: blank load transfection group as control group; P27-oe: over-expressed P27 in A549; P27-RNAi: Short Hairpin RNA on Expression of P27 in A549; CDK6-oe: over-expressed CDK6 in A549; CDK6-RNAi: Short Hairpin RNA on Expression of CDK6 in A549; CCND1-oe: over-expressed CCND1 in A549; CCND1-RNAi: Short Hairpin RNA on Expression of CCND1 in A549. Horizontal axis represents the hours after cell transfection. The vertical axis represents the cell number. (c) MTT reduction assay was used to reflect the proliferation at 0, 24, 36, and 48 h after changing the expression of related genes. Horizontal axis represents the hours after cell transfection. The vertical axis represents the OD value of cell proliferation. (d) Flow cytometry and MTT methods were used to detect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Transfection, shRNA, MTT Assay

    P27, CDK6 and CCND1 regulate the cell cycle in HTB182 cells. (a) Flow detection of cell cycle after changes to P27 expression. (b) Flow detection of cell cycle after changes to CDK6 expression. (c) Flow detection of cell cycle after changes to CCND1 expression. (d) Flow detection of cell cycle after changes to P27, CDK6, CCND1 expression.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: P27, CDK6 and CCND1 regulate the cell cycle in HTB182 cells. (a) Flow detection of cell cycle after changes to P27 expression. (b) Flow detection of cell cycle after changes to CDK6 expression. (c) Flow detection of cell cycle after changes to CCND1 expression. (d) Flow detection of cell cycle after changes to P27, CDK6, CCND1 expression.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Flow Cytometry, Expressing

    Expression of p27, CDK6 , and CCND1 in Drosophila , mice, and humans. (a) Twelve developmental stages from data selections: MM-AFFY-430 −2–0 Showing three measures of P27, CDK6 , and CCND1 in mice. The 12 stages were: prenatal_0–1, prenatal_2–4, prenatal_7–8.5, prenatal_9–11, prenatal_11.5–15, prenatal_16-18, postnatal_0, postnatal_1–3, postnatal_4–15, postnatal_16–63, adult_64-255, adult_256-9999. (b) Nine developmental stages from data selections: DM-AFFY-DG −2–0 Showing three measures of P27, CDK6 and CCND1 in Drosophila . The nine stages are: germ band elongation stage embryo, germ band retraction stage embryo, late stage embryo, first instar larval stage, second instar larval stage, third instar larval stage, prepupal development, pupal development, and adult development. (c) Detection of mRNA expression of p27, CDK6, CCND1 in six human organs. The organs included the stomach, lung, heart, kidney, adrenal gland, and intestine. (d) Analysis of p27, CDK6, CCND1 functions in humans. The functions of three genes involved in the regulation including cyclin-dependent protein serine/threonine activity, cyclin-dependent protein kinase activity, and G/S transition of mitotic cell cycle.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: Expression of p27, CDK6 , and CCND1 in Drosophila , mice, and humans. (a) Twelve developmental stages from data selections: MM-AFFY-430 −2–0 Showing three measures of P27, CDK6 , and CCND1 in mice. The 12 stages were: prenatal_0–1, prenatal_2–4, prenatal_7–8.5, prenatal_9–11, prenatal_11.5–15, prenatal_16-18, postnatal_0, postnatal_1–3, postnatal_4–15, postnatal_16–63, adult_64-255, adult_256-9999. (b) Nine developmental stages from data selections: DM-AFFY-DG −2–0 Showing three measures of P27, CDK6 and CCND1 in Drosophila . The nine stages are: germ band elongation stage embryo, germ band retraction stage embryo, late stage embryo, first instar larval stage, second instar larval stage, third instar larval stage, prepupal development, pupal development, and adult development. (c) Detection of mRNA expression of p27, CDK6, CCND1 in six human organs. The organs included the stomach, lung, heart, kidney, adrenal gland, and intestine. (d) Analysis of p27, CDK6, CCND1 functions in humans. The functions of three genes involved in the regulation including cyclin-dependent protein serine/threonine activity, cyclin-dependent protein kinase activity, and G/S transition of mitotic cell cycle.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Expressing, Mouse Assay, Activity Assay

    Expression of p27, CDK6, and CCND1 in lung cancer patients. (a) Expression of p27 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (b) Expression of CDK6 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (c) Expression of CCND1 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (d) Expression of p27 was detected in cancer tissues and paracancerous tissues of four lung squamous cell carcinoma patients by IHC. (e) Expression of p27 was detected in cancer tissues and paracancerous tissues of four lung adenocarcinoma patients by IHC.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: Expression of p27, CDK6, and CCND1 in lung cancer patients. (a) Expression of p27 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (b) Expression of CDK6 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (c) Expression of CCND1 in cancer tissues and the corresponding adjacent tissues of five lung cancer patients. (d) Expression of p27 was detected in cancer tissues and paracancerous tissues of four lung squamous cell carcinoma patients by IHC. (e) Expression of p27 was detected in cancer tissues and paracancerous tissues of four lung adenocarcinoma patients by IHC.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Expressing, Immunohistochemistry

    P27 affects the formation of CDK6/CCND1 complex. (a) qPCR was used to detect the expression of CDK6 and CCND1 after regulating the expression of P27 in A549 cells. (b) Co-immunoprecipitation and Western blotting were combined to examine the effects of p27 on formation of the CDK6/CCND1 complex after regulating the expression of P27 in A549 cells. (c) The distribution of CDK6 and CCND1 was detected by immunofluorescence after regulating the expression of p27 in A549 cells. (d) Phosphorylation of Rb was detected when the expression of p27 was changed in A549 and HTB182 cells.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: P27 affects the formation of CDK6/CCND1 complex. (a) qPCR was used to detect the expression of CDK6 and CCND1 after regulating the expression of P27 in A549 cells. (b) Co-immunoprecipitation and Western blotting were combined to examine the effects of p27 on formation of the CDK6/CCND1 complex after regulating the expression of P27 in A549 cells. (c) The distribution of CDK6 and CCND1 was detected by immunofluorescence after regulating the expression of p27 in A549 cells. (d) Phosphorylation of Rb was detected when the expression of p27 was changed in A549 and HTB182 cells.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

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

    P27, CDK6 and CCND1 affected the proliferation of HTB182 cells. (a) qPCR was used to detect the expression of p27, CDK6 and CCND1 after changing the expression levels of these genes. (b) The number of cells was detected by flow cytometry at 0, 24, 36, and 48 h after changing the expression of related genes. Control: blank load transfection group as control group; P27-oe: over-expressed P27 in HTB182; P27-RNAi: Short Hairpin RNA on Expression of P27 in HTB182; CDK6-oe: over-expressed CDK6 in HTB182; CDK6-RNAi: Short Hairpin RNA on Expression of CDK6 in HTB182; CCND1-oe: over-expressed CCND1 in HTB182; CCND1-RNAi: Short Hairpin RNA on Expression of CCND1 in HTB182. Horizontal axis represents the hours after cell transfection. The vertical axis represents the cell number. (c) MTT reduction assay was used to reflect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes. Horizontal axis represents the hours after cell transfection. The vertical axis represents the OD value of cell proliferation. (d) Flow cytometry and MTT methods were used to detect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes.

    Journal: Cell Cycle

    Article Title: p27 inhibits CDK6/CCND1 complex formation resulting in cell cycle arrest and inhibition of cell proliferation

    doi: 10.1080/15384101.2018.1526598

    Figure Lengend Snippet: P27, CDK6 and CCND1 affected the proliferation of HTB182 cells. (a) qPCR was used to detect the expression of p27, CDK6 and CCND1 after changing the expression levels of these genes. (b) The number of cells was detected by flow cytometry at 0, 24, 36, and 48 h after changing the expression of related genes. Control: blank load transfection group as control group; P27-oe: over-expressed P27 in HTB182; P27-RNAi: Short Hairpin RNA on Expression of P27 in HTB182; CDK6-oe: over-expressed CDK6 in HTB182; CDK6-RNAi: Short Hairpin RNA on Expression of CDK6 in HTB182; CCND1-oe: over-expressed CCND1 in HTB182; CCND1-RNAi: Short Hairpin RNA on Expression of CCND1 in HTB182. Horizontal axis represents the hours after cell transfection. The vertical axis represents the cell number. (c) MTT reduction assay was used to reflect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes. Horizontal axis represents the hours after cell transfection. The vertical axis represents the OD value of cell proliferation. (d) Flow cytometry and MTT methods were used to detect cell proliferation at 0, 24, 36, and 48 h after changing the expression of related genes.

    Article Snippet: All specimens were fixed in 4% formalin and embedded in paraffin, and cut into 4 μm thick sections, followed by immunohistochemistry using antibodies against p27 (CST).

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Transfection, shRNA, MTT Assay

    MLN8237 treatment activated the calpain pathway and induced apoptosis through p27 degradation and Bax cleavage in AGS cell line. a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f , g MLN8237-induced Ca 2+ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM ( f ). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: MLN8237 treatment activated the calpain pathway and induced apoptosis through p27 degradation and Bax cleavage in AGS cell line. a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f , g MLN8237-induced Ca 2+ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM ( f ). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Article Snippet: At least 20 μg of each protein sample was loaded for immunoblotting and tested by antibodies that against p27, p21, Skp2, c-PARP, c-caspase 3, c-caspase 9, cytochrome c, AURKA, AURKB (Cell Signaling Inc., Danvers, MA, USA), c-Bax, calpain 4 (Santa Cruz Biotechnology, Santa Cruz, USA), AIF (Epitomics, Hanghzou, China), HA-tag, Flag-tag, Actin (Cw Biotech, Shanghai, China).

    Techniques: Cell Culture, Concentration Assay, Incubation, Expressing, Immunoprecipitation, Labeling

    c-Bax localized to mitochondria and induced cytochrome c release and led to apoptosis. a Mitochondrial isolation to detect c-Bax distribution and cytochrome c release upon 200 nM MLN8237 treatment for 72 h in AGS. Mitochondrial isolation was conducted according to the manufacturer protocol and measured indicated protein expression in the various component of the cell lysate. b Location detection of EGFP-Bax and EGFP-ΔBax in 293 T. EGFP-Bax or EGFP-ΔBax was transfected into 293 T and photographed through a fluorescence microscope before incubated with mitotracker for 15 min at 37 °C. c Enhanced c-Bax localized to the mitochondrial after p27 silencing in 200 nM MLN8237-treated AGS cell line for 72 h. Cells were subjected to mitochondrial isolation and immunoblotting for quantification of mitochondrial c-Bax. d Augmented deterioration of the mitochondrial membrane potential (MMP) by p27 silencing in MLN8237-treated AGS cell line. AGS cells transfected with negative control or p27 siRNA were treated with 200 nM MLN8237 for 72 h, and subjected to JC-1 staining and FCM to detect MMP collapse. Error bars represent SD from three independent experiments. Representative microscopic images are shown. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: c-Bax localized to mitochondria and induced cytochrome c release and led to apoptosis. a Mitochondrial isolation to detect c-Bax distribution and cytochrome c release upon 200 nM MLN8237 treatment for 72 h in AGS. Mitochondrial isolation was conducted according to the manufacturer protocol and measured indicated protein expression in the various component of the cell lysate. b Location detection of EGFP-Bax and EGFP-ΔBax in 293 T. EGFP-Bax or EGFP-ΔBax was transfected into 293 T and photographed through a fluorescence microscope before incubated with mitotracker for 15 min at 37 °C. c Enhanced c-Bax localized to the mitochondrial after p27 silencing in 200 nM MLN8237-treated AGS cell line for 72 h. Cells were subjected to mitochondrial isolation and immunoblotting for quantification of mitochondrial c-Bax. d Augmented deterioration of the mitochondrial membrane potential (MMP) by p27 silencing in MLN8237-treated AGS cell line. AGS cells transfected with negative control or p27 siRNA were treated with 200 nM MLN8237 for 72 h, and subjected to JC-1 staining and FCM to detect MMP collapse. Error bars represent SD from three independent experiments. Representative microscopic images are shown. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Article Snippet: At least 20 μg of each protein sample was loaded for immunoblotting and tested by antibodies that against p27, p21, Skp2, c-PARP, c-caspase 3, c-caspase 9, cytochrome c, AURKA, AURKB (Cell Signaling Inc., Danvers, MA, USA), c-Bax, calpain 4 (Santa Cruz Biotechnology, Santa Cruz, USA), AIF (Epitomics, Hanghzou, China), HA-tag, Flag-tag, Actin (Cw Biotech, Shanghai, China).

    Techniques: Isolation, Expressing, Transfection, Fluorescence, Microscopy, Incubation, Negative Control, Staining

    The protective role of cytoplasmic p27 in MLN8237-induced Bax cleavage and apoptosis in AGS gastric cancer cell line. a p27 distribution in the cytoplasm of AGS. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according to manufacturer description. b p27 and phosphomimetic p27 mutant (T157D or T198D) overexpression inhibited MLN8237-induced Bax cleavage. AGS cells were transfected with Flag-tagged p27 or p27 mutant (T157D or T198D) and then treated with 200 nM MLN8237 for 72 h. c – f The combination of MLN8237 treatment and p27 silencing increased Bax cleavage. AGS cells were subjected to immunoblotting analysis c to determine the expression of the indicated proteins. MTS d was utilized to detect cell proliferation or FCM e , f to detect apoptosis level. 200 nM MLN8237 was incubated for 72 h in AGS. The mean and SDs of the plots were obtained from three wells within three independent MTS and apoptosis detection assays. The apoptosis staining experiment was performed independently three times. g Enhanced cytotoxicity of some chemotherapeutic agents after p27 silencing. 1 μM doxorubicin (Dox), 1 μM Hydroxycamptothecin (HPT), 0.3 μM MLN4924 or 10 μM oxaliplatin (Oxa) was added to culture after p27 silencing and subjected to MTS assay and immunoblotting at 72 h in AGS cell line. Error bars represent SD from three independent experiments. h The cytoplasmic localization of p27 in selected cancer cells. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according manufacturer description. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. * P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: The protective role of cytoplasmic p27 in MLN8237-induced Bax cleavage and apoptosis in AGS gastric cancer cell line. a p27 distribution in the cytoplasm of AGS. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according to manufacturer description. b p27 and phosphomimetic p27 mutant (T157D or T198D) overexpression inhibited MLN8237-induced Bax cleavage. AGS cells were transfected with Flag-tagged p27 or p27 mutant (T157D or T198D) and then treated with 200 nM MLN8237 for 72 h. c – f The combination of MLN8237 treatment and p27 silencing increased Bax cleavage. AGS cells were subjected to immunoblotting analysis c to determine the expression of the indicated proteins. MTS d was utilized to detect cell proliferation or FCM e , f to detect apoptosis level. 200 nM MLN8237 was incubated for 72 h in AGS. The mean and SDs of the plots were obtained from three wells within three independent MTS and apoptosis detection assays. The apoptosis staining experiment was performed independently three times. g Enhanced cytotoxicity of some chemotherapeutic agents after p27 silencing. 1 μM doxorubicin (Dox), 1 μM Hydroxycamptothecin (HPT), 0.3 μM MLN4924 or 10 μM oxaliplatin (Oxa) was added to culture after p27 silencing and subjected to MTS assay and immunoblotting at 72 h in AGS cell line. Error bars represent SD from three independent experiments. h The cytoplasmic localization of p27 in selected cancer cells. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according manufacturer description. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. * P

    Article Snippet: At least 20 μg of each protein sample was loaded for immunoblotting and tested by antibodies that against p27, p21, Skp2, c-PARP, c-caspase 3, c-caspase 9, cytochrome c, AURKA, AURKB (Cell Signaling Inc., Danvers, MA, USA), c-Bax, calpain 4 (Santa Cruz Biotechnology, Santa Cruz, USA), AIF (Epitomics, Hanghzou, China), HA-tag, Flag-tag, Actin (Cw Biotech, Shanghai, China).

    Techniques: Isolation, Mutagenesis, Over Expression, Transfection, Expressing, Incubation, Staining, MTS Assay

    Representative Sections of Oral SCC Demonstrating Positive Nuclear Immunostaining With Antibodies Against (A) p27 and (B) p53 (original magnification × 400) Arrow points to immunostained nuclei of neoplastic cells.

    Journal: Iranian Red Crescent Medical Journal

    Article Title: Status of p53 and p27KIP1 in Iranian Patients With Oral Squamous Cell Carcinoma

    doi: 10.5812/ircmj.19359

    Figure Lengend Snippet: Representative Sections of Oral SCC Demonstrating Positive Nuclear Immunostaining With Antibodies Against (A) p27 and (B) p53 (original magnification × 400) Arrow points to immunostained nuclei of neoplastic cells.

    Article Snippet: Incubation with monoclonal antibodies against p27 (IB4, Novocastra) and p53 (DO-7, Dako SA, Glostrup, Denmark) was carried out using dilutions of 1:20 and 1:50, respectively.

    Techniques: Immunostaining

    Kaplan-Meier Curve for Overall Survival in p27 Positive and Negative Oral SCC Subjects

    Journal: Iranian Red Crescent Medical Journal

    Article Title: Status of p53 and p27KIP1 in Iranian Patients With Oral Squamous Cell Carcinoma

    doi: 10.5812/ircmj.19359

    Figure Lengend Snippet: Kaplan-Meier Curve for Overall Survival in p27 Positive and Negative Oral SCC Subjects

    Article Snippet: Incubation with monoclonal antibodies against p27 (IB4, Novocastra) and p53 (DO-7, Dako SA, Glostrup, Denmark) was carried out using dilutions of 1:20 and 1:50, respectively.

    Techniques:

    XFC extract inhibits CDK4 and cyclin D3 in SKOV-3 ovarian cancer cells. Human ovarian clear cell carcinoma (ES-2) and human ovarian adenocarcinoma (SKOV-3) cells were cultured as described in Section 2 . Treatment with various concentrations of XFC was performed in serum-free media for 24 hours. Cell lysates were harvested and then processed for (a) SDS-PAGE and western blotting in order to assess the expression levels of Cdk2, CDk4, Cdk6, Cyclin D1, Cyclin D3, p27, and GAPDH. (b) Levels of expression were quantified using scanning densitometry.

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: Targeting Ovarian Cancer Cell Cytotoxic Drug Resistance Phenotype with Xanthium strumarium L. Extract

    doi: 10.1155/2019/6073019

    Figure Lengend Snippet: XFC extract inhibits CDK4 and cyclin D3 in SKOV-3 ovarian cancer cells. Human ovarian clear cell carcinoma (ES-2) and human ovarian adenocarcinoma (SKOV-3) cells were cultured as described in Section 2 . Treatment with various concentrations of XFC was performed in serum-free media for 24 hours. Cell lysates were harvested and then processed for (a) SDS-PAGE and western blotting in order to assess the expression levels of Cdk2, CDk4, Cdk6, Cyclin D1, Cyclin D3, p27, and GAPDH. (b) Levels of expression were quantified using scanning densitometry.

    Article Snippet: Polyclonal antibodies against Survivin, Nrf2, AKT, and phospho-AKT, PARP, Cyclin D1, Cyclin D3, Cdk2, Cdk4, Cdk6, and monoclonal antibody against p27 were from Cell Signaling Technology (Beverly, MA).

    Techniques: Cell Culture, SDS Page, Western Blot, Expressing

    p27 expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant

    Journal: Cancer genetics

    Article Title: Somatic alterations of CDKN1B are associated with small bowel neuroendocrine tumors

    doi: 10.1016/j.cancergen.2015.08.003

    Figure Lengend Snippet: p27 expression in primary tumor samples with missense mutations. (a) SBNET 162-1 demonstrates moderate to strong expression of p27 in the cytoplasm of the cell. The missense mutation in this sample was not predicted to be damaging per the Exome Variant

    Article Snippet: Immunohistochemistry was performed using a monoclonal antibody against p27 (clone SX53G8; Dako, Carpinteria, CA).

    Techniques: Expressing, Mutagenesis, Variant Assay

    p27 expression in tumors with somatic mutations

    Journal: Cancer genetics

    Article Title: Somatic alterations of CDKN1B are associated with small bowel neuroendocrine tumors

    doi: 10.1016/j.cancergen.2015.08.003

    Figure Lengend Snippet: p27 expression in tumors with somatic mutations

    Article Snippet: Immunohistochemistry was performed using a monoclonal antibody against p27 (clone SX53G8; Dako, Carpinteria, CA).

    Techniques: Expressing

    p27 expression in tumor samples with frameshift mutations (200× magnification). Positive staining is indicated by the brown color. Images a–c demonstrates p27 expression in a series of samples from the same patient (SBNET 318-1), with

    Journal: Cancer genetics

    Article Title: Somatic alterations of CDKN1B are associated with small bowel neuroendocrine tumors

    doi: 10.1016/j.cancergen.2015.08.003

    Figure Lengend Snippet: p27 expression in tumor samples with frameshift mutations (200× magnification). Positive staining is indicated by the brown color. Images a–c demonstrates p27 expression in a series of samples from the same patient (SBNET 318-1), with

    Article Snippet: Immunohistochemistry was performed using a monoclonal antibody against p27 (clone SX53G8; Dako, Carpinteria, CA).

    Techniques: Expressing, Staining

    Effects of resveratrol and/or TRAIL on Bcl-2 family members and cell cycle regulatory proteins. (A), Western blot analysis was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control, resveratrol and/or TRAIL treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of Bax and Bcl-2 positive cells in tumor cells (right panel). (B), Western blot analysis was performed to measure the expressions of p27 /KIP1  and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of p27 /KIP1  and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of p27 /KIP1  and cyclin D1 positive cells in tumor tissues (right panel).

    Journal: PLoS ONE

    Article Title: Resveratrol Enhances Antitumor Activity of TRAIL in Prostate Cancer Xenografts through Activation of FOXO Transcription Factor

    doi: 10.1371/journal.pone.0015627

    Figure Lengend Snippet: Effects of resveratrol and/or TRAIL on Bcl-2 family members and cell cycle regulatory proteins. (A), Western blot analysis was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control, resveratrol and/or TRAIL treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of Bax and Bcl-2 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of Bax and Bcl-2 positive cells in tumor cells (right panel). (B), Western blot analysis was performed to measure the expressions of p27 /KIP1 and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (left panel). Immunohistochemistry was performed to measure the expressions of p27 /KIP1 and cyclin D1 in tumor tissues derived from control and drug treated mice on week 6 (middle panel). Quantification of p27 /KIP1 and cyclin D1 positive cells in tumor tissues (right panel).

    Article Snippet: Antibodies against p27/KIP1 , phospho-FKHRL1, cyclin D1, MMP-2 and MMP-9 were purchased from Cell Signaling Technology, Inc. (Danvers, MA).

    Techniques: Western Blot, Derivative Assay, Mouse Assay, Immunohistochemistry

    Schematic representation of the signalling events involved. Postn binding to its receptor Itgavb3 leads to dephosphorylation of FAK inhibiting PI3K/Akt pathways that regulates the expression and activity of p27Kip1. This pathway maintains the HSCs in quiescent state.

    Journal: Nature Communications

    Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

    doi: 10.1038/ncomms13500

    Figure Lengend Snippet: Schematic representation of the signalling events involved. Postn binding to its receptor Itgavb3 leads to dephosphorylation of FAK inhibiting PI3K/Akt pathways that regulates the expression and activity of p27Kip1. This pathway maintains the HSCs in quiescent state.

    Article Snippet: Antibodies against p27kip1 and phospho-specific antibodies against FAK, PI3K, Akt and Src were purchased from Cell signaling Technology Inc. (Beverly, MA, USA), and used at a 1 μg ml−1 concentration.

    Techniques: Binding Assay, De-Phosphorylation Assay, Expressing, Activity Assay

    PI3K/Akt inhibition leading to p27kip1 expression inhibits HSC proliferation in vitro . ( a ) BM derived KLS cells cultured in serum-free medium with SCF+TPO without (ST) or with (STP) Postn were examined for expression of cell cycle regulatory genes by qRT-PCR ( n =6, t test: * P =0.03). ( b – e ) Lineage depleted BM cells cultured for 2 days in serum-free medium SCF+TPO without (ST) or with (STP) Postn, were stained for cell surface markers to identify HSCs along with intracellular staining for p27kip1 ( b ) and phosphorylated forms of PI3K ( c ), Akt ( d ) and FAK y397 ( e ) ( n =4). ( f , g ) BM derived KLS cells cultured in serum-free medium containing SCF and TPO for 2 days without (ST) or with (STP) Postn alone, or in combination with the FAK inhibitor PF-573228. After 5 days, cells were harvested and total cell expansion was measured ( f ). Harvested cells were stained for markers to identify LT-HSCs and the proportion of LT-HSCs in different culture conditions was examined ( n =6, t test: * P

    Journal: Nature Communications

    Article Title: Outside-in integrin signalling regulates haematopoietic stem cell function via Periostin-Itgav axis

    doi: 10.1038/ncomms13500

    Figure Lengend Snippet: PI3K/Akt inhibition leading to p27kip1 expression inhibits HSC proliferation in vitro . ( a ) BM derived KLS cells cultured in serum-free medium with SCF+TPO without (ST) or with (STP) Postn were examined for expression of cell cycle regulatory genes by qRT-PCR ( n =6, t test: * P =0.03). ( b – e ) Lineage depleted BM cells cultured for 2 days in serum-free medium SCF+TPO without (ST) or with (STP) Postn, were stained for cell surface markers to identify HSCs along with intracellular staining for p27kip1 ( b ) and phosphorylated forms of PI3K ( c ), Akt ( d ) and FAK y397 ( e ) ( n =4). ( f , g ) BM derived KLS cells cultured in serum-free medium containing SCF and TPO for 2 days without (ST) or with (STP) Postn alone, or in combination with the FAK inhibitor PF-573228. After 5 days, cells were harvested and total cell expansion was measured ( f ). Harvested cells were stained for markers to identify LT-HSCs and the proportion of LT-HSCs in different culture conditions was examined ( n =6, t test: * P

    Article Snippet: Antibodies against p27kip1 and phospho-specific antibodies against FAK, PI3K, Akt and Src were purchased from Cell signaling Technology Inc. (Beverly, MA, USA), and used at a 1 μg ml−1 concentration.

    Techniques: Inhibition, Expressing, In Vitro, Derivative Assay, Cell Culture, Quantitative RT-PCR, Staining

    bPTEN/SHIP −/− B cells proliferate in response to treatment with BAFF. (A) Purified splenic B cells from WT, bPTEN −/− , bSHIP −/− , or bPTEN/SHIP −/− mice were stimulated with anti-IgM F(ab’) 2 (α-IgM) in the presence or absence of BAFF or with APRIL as indicated. Proliferation was determined at 48 h by 3 H-thymidine incorporation. Y-axis values shown are ×10 −3 cpm. All assays were conducted in triplicates and SDs are shown as error bars. The results are representative of six experiments. (B) Western blots of protein lysates from WT, bPTEN −/− (P), bSHIP −/− (S), or bPTEN/SHIP −/− (PS) splenic B cells either freshly isolated (t = 0) or stimulated for 48 h, as indicated, were probed with anti-p27 kip1 , anti-cyclin D3, or anti-actin antibodies. (C) WT, bPTEN −/− , bSHIP −/− , or bPTEN/SHIP −/− B cells were stimulated with anti-IgM F(ab’) 2 (α-IgM), BAFF, or anti-IgM F(ab’) 2 (α-IgM) + BAFF for the indicated time points. Western blots were probed with antibodies recognizing the phosphorylated form of Akt (Ser 473). Total ERK1/2 was used as a loading control. For B and C, data are representative of n = 5 independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Coordinate suppression of B cell lymphoma by PTEN and SHIP phosphatases

    doi: 10.1084/jem.20091962

    Figure Lengend Snippet: bPTEN/SHIP −/− B cells proliferate in response to treatment with BAFF. (A) Purified splenic B cells from WT, bPTEN −/− , bSHIP −/− , or bPTEN/SHIP −/− mice were stimulated with anti-IgM F(ab’) 2 (α-IgM) in the presence or absence of BAFF or with APRIL as indicated. Proliferation was determined at 48 h by 3 H-thymidine incorporation. Y-axis values shown are ×10 −3 cpm. All assays were conducted in triplicates and SDs are shown as error bars. The results are representative of six experiments. (B) Western blots of protein lysates from WT, bPTEN −/− (P), bSHIP −/− (S), or bPTEN/SHIP −/− (PS) splenic B cells either freshly isolated (t = 0) or stimulated for 48 h, as indicated, were probed with anti-p27 kip1 , anti-cyclin D3, or anti-actin antibodies. (C) WT, bPTEN −/− , bSHIP −/− , or bPTEN/SHIP −/− B cells were stimulated with anti-IgM F(ab’) 2 (α-IgM), BAFF, or anti-IgM F(ab’) 2 (α-IgM) + BAFF for the indicated time points. Western blots were probed with antibodies recognizing the phosphorylated form of Akt (Ser 473). Total ERK1/2 was used as a loading control. For B and C, data are representative of n = 5 independent experiments.

    Article Snippet: Antibodies raised against p27kip1 , cyclin D3, actin, phospho-Akt (S473), total Akt, phospho-GSK3β (S9), total ERK1/2, and Bim were obtained from Cell Signaling Technology.

    Techniques: Purification, Mouse Assay, Western Blot, Isolation

    Transgenic expression of p27 driven by the aP2 promoter suppressed cell proliferation in BAT. ( A – C ) Representative images of BAT sections from Tg mice show a lower expression of the cell proliferation markers, PCNA and Ki67, compared to WT mice ( A ). Quantification of PCNA-positive cells ( B ) and western blot analyses ( C ) in the BAT confirmed decreased expression of proliferative markers but there were no essential differences with respect to the apoptosis-related protein (n = 5 per group, Student’s t-test, *p

    Journal: Scientific Reports

    Article Title: Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

    doi: 10.1038/s41598-017-07206-8

    Figure Lengend Snippet: Transgenic expression of p27 driven by the aP2 promoter suppressed cell proliferation in BAT. ( A – C ) Representative images of BAT sections from Tg mice show a lower expression of the cell proliferation markers, PCNA and Ki67, compared to WT mice ( A ). Quantification of PCNA-positive cells ( B ) and western blot analyses ( C ) in the BAT confirmed decreased expression of proliferative markers but there were no essential differences with respect to the apoptosis-related protein (n = 5 per group, Student’s t-test, *p

    Article Snippet: Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA).

    Techniques: Transgenic Assay, Expressing, Mouse Assay, Western Blot

    Expression of p27 has no effect on adipocyte differentiation. ( A – D ) Mouse embryonic fibroblasts (MEFs) prepared from WT or aP2-p27 Tg mice embryos at day 16.5 of gestation were cultured and differentiated into adipocytes by treatment with 0.5 mM IBMX and1 µM Dex for the first two days, and subsequently with 10 µg/ml insulin, 50 nM T3, and 10 µM Tro for six days. Assessment of the expression of aP2 during differentiation ( A , C ) and cellular lipid staining with Oil Red O at Day 8 ( B ) showed that there were no differences in adipocyte differentiation between the MEFs of WT and Tg mice. Conversely, the expression of UCP1 mRNA ( C ) and the number of UCP1-expressing cells ( D ) were significantly decreased in MEFs from Tg mice, compared to WT mice, and accompanied by increased p27 expression ( A ) (n = 4 per group, Student’s t-test, *p

    Journal: Scientific Reports

    Article Title: Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

    doi: 10.1038/s41598-017-07206-8

    Figure Lengend Snippet: Expression of p27 has no effect on adipocyte differentiation. ( A – D ) Mouse embryonic fibroblasts (MEFs) prepared from WT or aP2-p27 Tg mice embryos at day 16.5 of gestation were cultured and differentiated into adipocytes by treatment with 0.5 mM IBMX and1 µM Dex for the first two days, and subsequently with 10 µg/ml insulin, 50 nM T3, and 10 µM Tro for six days. Assessment of the expression of aP2 during differentiation ( A , C ) and cellular lipid staining with Oil Red O at Day 8 ( B ) showed that there were no differences in adipocyte differentiation between the MEFs of WT and Tg mice. Conversely, the expression of UCP1 mRNA ( C ) and the number of UCP1-expressing cells ( D ) were significantly decreased in MEFs from Tg mice, compared to WT mice, and accompanied by increased p27 expression ( A ) (n = 4 per group, Student’s t-test, *p

    Article Snippet: Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Mouse Assay, Cell Culture, Staining

    Transgenic expression of p27 driven by the aP2 promoter did not affect WAT development but suppressed BAT development. ( A ) There were no differences in body weight between 12-week-old WT and aP2-p27 Tg mice. ( B – D ) Tissue weights ( C ) and representative examples of the gross ( B ) and histological ( D ) appearance show that inguinal and peri-gonadal WAT (I-WAT and G-WAT) in Tg mice is highly similar to I-WAT and G-WAT in WT mice. Conversely, interscapular BAT in Tg mice was reduced in size and volume but had normal histological features, compared to BAT in WT mice (n = 5 per group, Student’s t-test, *p

    Journal: Scientific Reports

    Article Title: Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

    doi: 10.1038/s41598-017-07206-8

    Figure Lengend Snippet: Transgenic expression of p27 driven by the aP2 promoter did not affect WAT development but suppressed BAT development. ( A ) There were no differences in body weight between 12-week-old WT and aP2-p27 Tg mice. ( B – D ) Tissue weights ( C ) and representative examples of the gross ( B ) and histological ( D ) appearance show that inguinal and peri-gonadal WAT (I-WAT and G-WAT) in Tg mice is highly similar to I-WAT and G-WAT in WT mice. Conversely, interscapular BAT in Tg mice was reduced in size and volume but had normal histological features, compared to BAT in WT mice (n = 5 per group, Student’s t-test, *p

    Article Snippet: Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA).

    Techniques: Transgenic Assay, Expressing, Mouse Assay

    Transgenic expression of p27 driven by the aP2 promoter did not affect the generation and function of beige adipocytes. Mice acclimated to a room temperature of 28 °C were injected with saline or CL316,243 (CL; 1 mg/kg/day, s.c.) for 14 days. ( A and B ) Representative histological images ( A ) and measurement of the UCP1 content per inguinal-WAT (I-WAT) ( B ) show that there were no differences between WT and Tg mice with regard to the induction of beige adipocytes by CL. ( C – E ) Analysis of adipocyte fractions isolated from the I-WAT of WT and Tg mice after CL treatment showed the presence of a similar number of multilocular beige adipocytes ( C ), similar levels of UCP1 ( D ), and consumption of a similar amount of oxygen in either the basal or norepinephrine-induced state ( E ). ( F ) Chronic CL treatment failed to increase levels of UCP1 per BAT in Tg mice. (n = 6 per group, one-way ANOVA followed by the Tukey–Kramer post-hoc test, different letters indicate significant differences between groups, p

    Journal: Scientific Reports

    Article Title: Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

    doi: 10.1038/s41598-017-07206-8

    Figure Lengend Snippet: Transgenic expression of p27 driven by the aP2 promoter did not affect the generation and function of beige adipocytes. Mice acclimated to a room temperature of 28 °C were injected with saline or CL316,243 (CL; 1 mg/kg/day, s.c.) for 14 days. ( A and B ) Representative histological images ( A ) and measurement of the UCP1 content per inguinal-WAT (I-WAT) ( B ) show that there were no differences between WT and Tg mice with regard to the induction of beige adipocytes by CL. ( C – E ) Analysis of adipocyte fractions isolated from the I-WAT of WT and Tg mice after CL treatment showed the presence of a similar number of multilocular beige adipocytes ( C ), similar levels of UCP1 ( D ), and consumption of a similar amount of oxygen in either the basal or norepinephrine-induced state ( E ). ( F ) Chronic CL treatment failed to increase levels of UCP1 per BAT in Tg mice. (n = 6 per group, one-way ANOVA followed by the Tukey–Kramer post-hoc test, different letters indicate significant differences between groups, p

    Article Snippet: Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA).

    Techniques: Transgenic Assay, Expressing, Mouse Assay, Injection, Isolation

    aP2-p27 transgenic mice had a lower amount of UCP1 and an attenuated thermogenic response to cold exposure or β3-AR agonism. ( A ) The total amounts of DNA per BAT in Tg mice were reduced compared to those in WT mice (n = 5 per group, Student’s t-test, *p

    Journal: Scientific Reports

    Article Title: Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice

    doi: 10.1038/s41598-017-07206-8

    Figure Lengend Snippet: aP2-p27 transgenic mice had a lower amount of UCP1 and an attenuated thermogenic response to cold exposure or β3-AR agonism. ( A ) The total amounts of DNA per BAT in Tg mice were reduced compared to those in WT mice (n = 5 per group, Student’s t-test, *p

    Article Snippet: Antibodies against aP2, caspase-9, and the phosphorylated form of Rb were purchased from Cell Signaling Technology (Beverly, MA, USA); those against β-actin, p57, and a-Tubulin were from Sigma-Aldrich; those against p27 and Rb were from BD Biosciences (San Jose, CA, USA); that against PCNA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and those against Ki67 and MCT1 were from Abcam (Cambridge, MA, USA).

    Techniques: Transgenic Assay, Mouse Assay

    Increased expression of p27 in serum-starved Rb1 G/G MEFs. (A) Fibroblast cells of the indicated genotypes were serum starved for 60 h and pulse-labeled with BrdU for 2 h, followed by staining for BrdU incorporation. The proportion of cells incorporating

    Journal: Molecular and Cellular Biology

    Article Title: Interchangeable Roles for E2F Transcriptional Repression by the Retinoblastoma Protein and p27KIP1–Cyclin-Dependent Kinase Regulation in Cell Cycle Control and Tumor Suppression

    doi: 10.1128/MCB.00561-16

    Figure Lengend Snippet: Increased expression of p27 in serum-starved Rb1 G/G MEFs. (A) Fibroblast cells of the indicated genotypes were serum starved for 60 h and pulse-labeled with BrdU for 2 h, followed by staining for BrdU incorporation. The proportion of cells incorporating

    Article Snippet: Antibodies raised against p27 (C-19; sc-528) and histone H3 (ab70550) were used for Western blotting. pRB-containing complexes were immunoprecipitated from whole-cell extracts using anti-E2F3 C-18 (Santa Cruz) bound to G-Sepharose beads (GE Healthcare).

    Techniques: Expressing, Labeling, Staining, BrdU Incorporation Assay

    Knockdown of DICER prolongs the G1/S transition through up-regulating p21 Waf1/Cip1 and p27/ Kip1 . ( A ) The number of human cells (MRC5SV, HeLa, ATM −/− , M059J and 180BRM) was counted at 72 h after the cells were treated with CtRNA or siDICER. The data were obtained from two independent experiments. ( B ) HeLa cells were collected at 60 h after treatment with CtRNA or siDICER. HeLa cells in the G1 and S phases were detected by combining dye from PI, BrdU labeling and flow cytometry as described in the Supplementary Materials. Similar results were obtained from three independent experiments that the authors carried out. ( C ) Immunoblot detection was performed from MRC5SV and HeLa cells treated with siDICER for 60 h. Similar data were obtained from two independent experiments that the authors carried out. ( D ) HeLa cells were treated with siDICER with or without siRNA against p21 or/and p27 (si-p21, si-p27 or si-p21/si-p27) for 60 h, and the cells were collected for immunoblot detection. Similar data were obtained from two independent experiments. ( E ) The effects of si-p21/si-p27 on the G1-S transition in the DICER knockdowns. HeLa cells were collected for detecting G1 and S phase cell distribution as described in B. Similar results were obtained from three independent experiments that the authors carried out.

    Journal: Nucleic Acids Research

    Article Title: DICER-dependent biogenesis of let-7 miRNAs affects human cell response to DNA damage via targeting p21/p27

    doi: 10.1093/nar/gku1368

    Figure Lengend Snippet: Knockdown of DICER prolongs the G1/S transition through up-regulating p21 Waf1/Cip1 and p27/ Kip1 . ( A ) The number of human cells (MRC5SV, HeLa, ATM −/− , M059J and 180BRM) was counted at 72 h after the cells were treated with CtRNA or siDICER. The data were obtained from two independent experiments. ( B ) HeLa cells were collected at 60 h after treatment with CtRNA or siDICER. HeLa cells in the G1 and S phases were detected by combining dye from PI, BrdU labeling and flow cytometry as described in the Supplementary Materials. Similar results were obtained from three independent experiments that the authors carried out. ( C ) Immunoblot detection was performed from MRC5SV and HeLa cells treated with siDICER for 60 h. Similar data were obtained from two independent experiments that the authors carried out. ( D ) HeLa cells were treated with siDICER with or without siRNA against p21 or/and p27 (si-p21, si-p27 or si-p21/si-p27) for 60 h, and the cells were collected for immunoblot detection. Similar data were obtained from two independent experiments. ( E ) The effects of si-p21/si-p27 on the G1-S transition in the DICER knockdowns. HeLa cells were collected for detecting G1 and S phase cell distribution as described in B. Similar results were obtained from three independent experiments that the authors carried out.

    Article Snippet: The antibodies against human DICER, DNA-PKcs, Ku70, Lig4, XRCC4, p27/Kip1 (also against mouse p27/Kip1 ), CHK1, CHK2, Rad51, Rad54, Cyclin E, Cyclin A, HA, Actin, the mouse DICER and p21Waf1/Cip1 were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Labeling, Flow Cytometry, Cytometry

    Knockdown of DICER-upregulated p21 waf1/Cip1 and p27/ Kip1 is due to the reduction of biogenesis of let-7 . ( A ) Fold reduction of the human let-7 family members from the microArray of miRNAs from MRC5SV cells treated with siDICER compared with the cells treated with CtRNA. Whole RNA was extracted from the cells and 1 μg of RNA was used for miRNA microarray by LC Sciences Inc. ( B ) The effects of let-7b mimics on p21 and p27 expression in the cells with or without DICER knockdown. MRC5SV and HeLa cells were treated with control RNA or siDICER with or without let-7 mimics for 60 h and then the cells were collected for immunoblot detection. ( C ) Description of potential let-7b binding sites at the 3′-UTR of p21 (left) or p27 (right). ( D ) Relative luciferase activities were detected in 293FT cells treated with CtRNA or let-7 mimics at 48 h after transfection with the luciferase reporter vector containing either the wild type 3′-UTR of p21 or p27 (WT) or the 3′-UTR mutated (MT) at the potential binding site for let-7 (p21: mutated CTACCT to GATGGA; p27: mutated CTACCT to GATGGA). The data presented are the mean ± SD from three independent experiments that the authors carried out, ** P

    Journal: Nucleic Acids Research

    Article Title: DICER-dependent biogenesis of let-7 miRNAs affects human cell response to DNA damage via targeting p21/p27

    doi: 10.1093/nar/gku1368

    Figure Lengend Snippet: Knockdown of DICER-upregulated p21 waf1/Cip1 and p27/ Kip1 is due to the reduction of biogenesis of let-7 . ( A ) Fold reduction of the human let-7 family members from the microArray of miRNAs from MRC5SV cells treated with siDICER compared with the cells treated with CtRNA. Whole RNA was extracted from the cells and 1 μg of RNA was used for miRNA microarray by LC Sciences Inc. ( B ) The effects of let-7b mimics on p21 and p27 expression in the cells with or without DICER knockdown. MRC5SV and HeLa cells were treated with control RNA or siDICER with or without let-7 mimics for 60 h and then the cells were collected for immunoblot detection. ( C ) Description of potential let-7b binding sites at the 3′-UTR of p21 (left) or p27 (right). ( D ) Relative luciferase activities were detected in 293FT cells treated with CtRNA or let-7 mimics at 48 h after transfection with the luciferase reporter vector containing either the wild type 3′-UTR of p21 or p27 (WT) or the 3′-UTR mutated (MT) at the potential binding site for let-7 (p21: mutated CTACCT to GATGGA; p27: mutated CTACCT to GATGGA). The data presented are the mean ± SD from three independent experiments that the authors carried out, ** P

    Article Snippet: The antibodies against human DICER, DNA-PKcs, Ku70, Lig4, XRCC4, p27/Kip1 (also against mouse p27/Kip1 ), CHK1, CHK2, Rad51, Rad54, Cyclin E, Cyclin A, HA, Actin, the mouse DICER and p21Waf1/Cip1 were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Microarray, Expressing, Binding Assay, Luciferase, Transfection, Plasmid Preparation

    Simplified representation of Type I and Type II circuits. The master transcription factors are cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4), and p27 is the target gene. Inside each circuit, → indicates transcription activation and ⊥ indicates post-transcriptional repression.

    Journal: Oncology Letters

    Article Title: The regulatory loop of COMP1 and HNF-4-miR-150-p27 in various signaling pathways

    doi: 10.3892/ol.2014.2643

    Figure Lengend Snippet: Simplified representation of Type I and Type II circuits. The master transcription factors are cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4), and p27 is the target gene. Inside each circuit, → indicates transcription activation and ⊥ indicates post-transcriptional repression.

    Article Snippet: Western blotting The expression levels of p27 protein were quantified via western blot analysis of the whole cell extracts using a monoclonal rabbit anti-human antibody against p27 (1:1,000; 3688, Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Activation Assay

    Graphical representation of the network consisting of cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4)-microRNA (miR)-150-p27, including certain important signaling pathways. Arrows indicate activation. Lines with blunt ends indicate post-transcriptional repression. Lines with an oval on the end indicate the uncertain transcription regulation of COMP1 and HNF-4 to miR-150 or p27, which may be active or supressive.

    Journal: Oncology Letters

    Article Title: The regulatory loop of COMP1 and HNF-4-miR-150-p27 in various signaling pathways

    doi: 10.3892/ol.2014.2643

    Figure Lengend Snippet: Graphical representation of the network consisting of cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4)-microRNA (miR)-150-p27, including certain important signaling pathways. Arrows indicate activation. Lines with blunt ends indicate post-transcriptional repression. Lines with an oval on the end indicate the uncertain transcription regulation of COMP1 and HNF-4 to miR-150 or p27, which may be active or supressive.

    Article Snippet: Western blotting The expression levels of p27 protein were quantified via western blot analysis of the whole cell extracts using a monoclonal rabbit anti-human antibody against p27 (1:1,000; 3688, Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Activation Assay

    Identification of conserved microRNA (miR)-150 binding sites within the p27 3′-UTR, and regulation of p27 expression by miR-150 at the mRNA and protein levels. (A) Diagram of the luciferase reporter plasmid carrying the firefly luciferase coding sequence fused to the human p27 3′-untranslated region (UTR) with wild-type miR-150 binding sites. (B) Direct recognition of the p27 3′-UTR by miR-150. Firefly luciferase reporters containing the p27 3′-UTR were cotransfected into MCF-7 cells with scrambled ncRNA, pre-miR-150 or anti-miR-150. At 24 h post-transfection, the cells were assayed using a luciferase assay kit. The y-axis shows arbitrary units representing relative luciferase activity. (C and D) Overexpression or knockdown of miR-150. MCF-7 cells were seeded in six-well plates and transfected the following day with scrambled ncRNA, pre-miR-150 or anti-miR-150 (200 pmol each) using Lipofectamine 2000. miR-150 levels were evaluated by reverse transcription quantitative polymerase chain reaction at 24 h post-transfection. For comparison, the expression levels of miR-150 in ncRNA-transfected cells were arbitrarily set as 1. The y-axis shows arbitrary units representing relative miR-150 expression levels. (E) Western blot analysis of the p27 protein levels in THP1 cells that were untreated or treated with anti-miR-150 at 48 h post-transfection. The results are presented as the mean ± SD of three independent experiments.

    Journal: Oncology Letters

    Article Title: The regulatory loop of COMP1 and HNF-4-miR-150-p27 in various signaling pathways

    doi: 10.3892/ol.2014.2643

    Figure Lengend Snippet: Identification of conserved microRNA (miR)-150 binding sites within the p27 3′-UTR, and regulation of p27 expression by miR-150 at the mRNA and protein levels. (A) Diagram of the luciferase reporter plasmid carrying the firefly luciferase coding sequence fused to the human p27 3′-untranslated region (UTR) with wild-type miR-150 binding sites. (B) Direct recognition of the p27 3′-UTR by miR-150. Firefly luciferase reporters containing the p27 3′-UTR were cotransfected into MCF-7 cells with scrambled ncRNA, pre-miR-150 or anti-miR-150. At 24 h post-transfection, the cells were assayed using a luciferase assay kit. The y-axis shows arbitrary units representing relative luciferase activity. (C and D) Overexpression or knockdown of miR-150. MCF-7 cells were seeded in six-well plates and transfected the following day with scrambled ncRNA, pre-miR-150 or anti-miR-150 (200 pmol each) using Lipofectamine 2000. miR-150 levels were evaluated by reverse transcription quantitative polymerase chain reaction at 24 h post-transfection. For comparison, the expression levels of miR-150 in ncRNA-transfected cells were arbitrarily set as 1. The y-axis shows arbitrary units representing relative miR-150 expression levels. (E) Western blot analysis of the p27 protein levels in THP1 cells that were untreated or treated with anti-miR-150 at 48 h post-transfection. The results are presented as the mean ± SD of three independent experiments.

    Article Snippet: Western blotting The expression levels of p27 protein were quantified via western blot analysis of the whole cell extracts using a monoclonal rabbit anti-human antibody against p27 (1:1,000; 3688, Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Binding Assay, Expressing, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Over Expression, Real-time Polymerase Chain Reaction, Western Blot