Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 4. Effects of estradiol on cyclin protein expression. The experimental design was described in Fig. 2. Representative Western blots are shown for G1 phase cyclins (D1, D3, E) (A) and S/G2M phase cyclins (A, B1) (B). Controls for cyclin E are as described in Fig. 2.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 5. Estrogen induction of cyclin D1 and cyclin D3 mRNA expression. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, total cellular RNA was harvested. A, representative North- ern blots are shown for cyclin D1 and D3 mRNA from estradiol-treated and control cells. Arrows indicate the 4.5- and 1.5-kb cyclin D1 transcripts. B, graphical pres- entation of temporal changes in mRNA (open symbols) and protein (solid symbols, mean of two experiments) for cyclin D1 (left) and cyclin D3 (right).

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 8. Activation of Cdk4 and Cdk2 following estrogen treatment. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immunoprecipitated with antibodies to either Cdk4, cyclin E, or Cdk2, and then the kinase activity of the immunoprecipitates was determined by phosphorylation of GST-pRB773–923

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Immunoprecipitation, Activity Assay, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 9. Composition of cyclin D1-as- sociated and cyclin E-associated complexes. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immuno- precipitated with anti-cyclin D1 anti- serum or anti-cyclin E antibodies, and then these immunoprecipitates were sep- arated by SDS-PAGE and transferred to nitrocellulose membranes. A, cyclin D1 antiserum immunoprecipitates. The same filter was sequentially Western blotted for cyclin D1, Cdk4, p21, and p27. A rep- resentative blot is shown for each. B, rel- ative levels of cyclin D1 (E), Cdk4 (G), p21 (M), and p27 (f) were determined by den- sitometry and are expressed relative to the vehicle treated controls. Points repre- sent the mean of two separate experi- ments. C, cyclin E immunoprecipitates. The same filter was sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Control, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 10. Estrogen decreases inhibi- tory activity toward cyclin E-Cdk2. A, lysates were prepared from cells that were pretreated with antiestrogen and then treated with E2 (1) or vehicle (2). Active cyclin E-Cdk2 complexes that had been prepared from baculovirus-infected Sf9 cells were incubated with either lysis buffer only (labeled input) or with cell lysates. Lysates were also immunode- pleted with either anti-p27 antibodies, anti-p21 antibodies, or both and then in- cubated with recombinant cyclin E-Cdk2 complexes. Recombinant cyclin E-Cdk2 complexes were then recovered and as- sayed for histone (H1) kinase activity. The percentage of the input activity, de- fined as 100%, is also shown numerically below the autoradiograph. The same sam- ples were electrophoresed on a duplicate SDS-PAGE gel and then Western blotted for p21 and p27 to assess the binding of these proteins to recombinant cyclin E- Cdk2. B, the experiment described for panel A was repeated using either boiled lysis buffer (labeled input) or boiled ly- sates from cells treated with estradiol (1) or vehicle (2). C, MCF-7 cells were pre- treated with 10 nM ICI 182780 for 48 h and then treated with 100 nM estradiol or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were pre- pared and assayed for cyclin E-Cdk2 in- hibitory activity as described for panel A.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activity Assay, Infection, Incubation, Lysis, Labeling, Recombinant, Autoradiography, SDS Page, Western Blot, Binding Assay, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 11. Cyclin E-Cdk2 activation is accompanied by loss of CDK inhibitor association and Cdk2 Thr-160 phosphorylation. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. Whole cell lysates were prepared either 8 h after estrogen (8 h E2) or from control cells (ICI 182780). Lysates were then fractionated on a Superose 12 gel filtration column. A, fractions were precipitated with acetone and Western blot- ted for cyclin E (top panels) or assayed for cyclin E-Cdk2 histone (H1) kinase activity (bottom panel). The relative levels of cyclin E protein (E, G) and cyclin E-Cdk2 activity (M, f) in lysates from antiestrogen- pretreated cells and cells treated with estradiol for 8 h were determined by densitometry and are represented graphically. The elution of mark- ers of known molecular weight (ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) are indicated at the top of the graph. B, fractions 19 and 24 from the 8 h E2 lysate were immunoprecipitated with an anti- cyclin E antibody, and the immunoprecipitates were electrophoresed on a SDS-PAGE gel and transferred to a nitrocellulose filter. The same filter was then sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Control, Filtration, Western Blot, Activity Assay, Molecular Weight, Immunoprecipitation, SDS Page

Journal: Cancer biology & therapy

Article Title: STAT3 inhibitor in combination with irradiation significantly inhibits cell viability, cell migration, invasion and tumorsphere growth of human medulloblastoma cells.

doi: 10.1080/15384047.2021.1951573

Figure Lengend Snippet: Figure 7. LLY17 or LLL12B combined with irradiation inhibited STAT3 targets and induced cell apoptosis protein in human medulloblastoma cells. Non-irradiated or irradiated (4 Gy) human medulloblastoma D283, D425, UW288, and UW426 cells were treated with LLY17 (a), LLL12B (b) or DMSO overnight. Cells were harvested and analyzed by Western blot. The expression levels of P-STAT3 (Y705), STAT3, Cyclin D1, Survivin and cleaved Caspase-3 were determined. GAPDH was used as a protein loading control.

Article Snippet: The primary antibodies for detecting P-STAT3 (Y705), STAT3, N-cadherin, SLUG, Cyclin D1, Survivin, cleaved Caspase-3, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Irradiation, Western Blot, Expressing, Control

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 2. Effects of si-YB-1 on cell cycle progression in human OS cell lines. (A) Representative cell cycle profile of MG63 and MNNG cells transfected with si-Ctr, si-YB-1#1, or si-YB-1#2. Cells were treated with siRNA for 72 h, and then detached from the substratum by limited trypsin digestion, and a single-cell suspension was used for propidium iodide staining. DNA content in single cells was measured by flow cytometry. (B) Increased G1/G0 and decreased S phase DNA content by YB-1 knockdown in MG63 and MNNG cell lines. Experiments were performed in triplicate, and data are expressed as the mean±s.d. *Po0.05.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Suspension, Staining, Flow Cytometry, Knockdown

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 3. Silencing the YB-1 gene in OS cells modulates cell cycle-related genes. (A) Effect of YB-1 knockdown on expression of cyclin D1, cyclin E, cyclin A, YB-1, and actin protein was analysed by immunoblotting. Cells were incubated with 50 nmol l 1 of si-Ctr or si-YB-1#1 for 48 h, and lysates were prepared. (B) Effect of YB-1 knockdown on cyclin D1 and cyclin A mRNA expression. Experiments were performed in triplicate, and data are expressed as the mean±s.d. *Po0.05. (C) Chromatin immunoprecipitation of cyclin D1 gene promoters using YB-1 antibody. Chromatin from MG63 and MNNG cell lines were cross-linked to fix bound proteins to the DNA. Cells were lysed and the chromatin was incubated with a YB-1 antibody to immunoprecipitate promoters bound by YB-1. Polymerase chain reaction was then performed to amplify promoter fragments to known to YB-1 bound. Input ¼ DNA before immunoprecipitation; IgG ¼ ChIP with the IgG-negative control antibody; Ab ¼ YB-1 antibody. Figure shows typical results obtained from at least three independent experiments. (D) Effect of silencing of the YB-1 gene on expression of E2F1, and YB-1 protein was analysed by immunoblotting. Actin was used for internal normalisation. Effect of YB-1 knockdown on E2F1 mRNA expression. Experiments were performed in triplicate, and data are expressed as the mean±s.d. NS ¼ nonsignificant.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Expressing, Western Blot, Incubation, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Negative Control

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 4. Effects of si-cyclin D1 on cell proliferation and cell cycle progression in human OS cell lines. (A) Growth curve of MG63 and MNNG cells transfected with si-Ctr or si-cyclin D1 monitored up to 96 h post-transfection. Experiments were performed in triplicate, and data are expressed as the mean±s.d. *Po0.05. (B) Representative cell cycle profile of MG63 and MNNG cells transfected with si-Ctr or si-cyclin D1. (C) Effect of silencing of the cyclin D1 gene on expression of cyclin D1, cyclin A, and YB-1 protein was analysed by immunoblotting. Actin was used for internal normalisation.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Western Blot

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 5. Effects of si-cyclin A on cell proliferation and cell cycle progression in human OS cell lines. (A) Growth curve of MG63 and MNNG cells transfected with si-Ctr or si-cyclin A monitored up to 96 h post-transfection. Experiments were performed in triplicate, and data are expressed as the mean±s.d. *Po0.05, **Po0.001. (B) Representative cell cycle profile of MG63 and MNNG cells transfected with si-Ctr or si-cyclin A. (C) Effect of silencing the cyclin A gene on expression of cyclin A, cyclin D1, and YB-1 protein was analysed by immunoblotting. Actin was used for internal normalisation.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Western Blot

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 6. Effects of overexpression of YB-1 and cyclin D1 on si-YB-1-induced suppression of cell proliferation. (A) MG63 and MNNG cells were transfected with si-Ctr, si-YB-1#1 (50 pmol l 1) and expression vectors of YB-1 (500 ng and 2 mg) and cyclin D1 for 48 h. The cells were harvested with trypsin and counted. Experiments were performed in triplicate and data are expressed as the mean±s.d. *Po0.05. (B) MG63 cells were transfected with si-Ctr, si-YB-1#1 (50 pmol l 1) and expression vectors of YB-1 (500 ng and 2 mg) and cyclin D1 for 48 h and whole-cell extracts were subjected to SDS–PAGE, and western blot analysis was done with corresponding antibodies. Actin was used for internal normalisation. NS ¼ nonsignificant.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Over Expression, Transfection, Expressing, SDS Page, Western Blot

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 7. Inhibition of tumour growth by si-YB-1 with atelocollagen in the MNNG xenograft model. (A) Tumour growth curves after treatment with si-YB-1#1 or si-Ctr with atelocollagen. Each therapeutic reagent was injected into the tumours on days 0, 7, and 14 (arrows). Data are expressed as the mean±s.d. (n ¼ 12 for YB-1, n ¼ 10 for Ctr). **Po0.01, when si-YB-1#1 was compared with si-Ctr. (B) Levels of YB-1, cyclin D1, and cyclin A in tumours were analysed by immunoblotting. Actin was used for internal normalisation. (C) Representative micrographs of haematoxylin eosin staining and immunohistochemical detection of YB-1, cyclin D1, cyclin A, and MIB-1 in tumours treated with si-YB-1#1(left) or si-Ctr (right). Scale bar; 20 mm (D and E). The number of cells expressing YB-1 (D) and MIB-1 (E) was scored in five independent areas. The percentage of YB-1- or MIB-1-positive cells was then calculated. Data are expressed as the mean±s.d. **Po0.01 si-YB-1#1 vs si-Ctr.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Injection, Western Blot, Staining, Immunohistochemical staining, Expressing

Journal: British journal of cancer

Article Title: Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

doi: 10.1038/bjc.2012.579

Figure Lengend Snippet: Figure 8. Haematoxylin eosin and immunohistochemical staining of human OS sections. Representative staining of YB-1, cyclin D1, cyclin A, and MIB-1 in OS samples. Paraffin sections were stained with haematoxylin eosin and immunohistochemically stained using anti-YB- 1, anti-cyclin D1, anti-cyclin A, and anti-YB-1 antibodies, then were visualised using the diaminobenzidene substrate system. Counterstaining was then performed using diluted haematoxylin. In case 38 (YB-1 nuclear expression positive, died of disease), high levels of cyclin D1 (X10%) and cyclin A (X40%) expression were evident, whereas in case 12 (YB-1 nuclear expression negative, continuously disease free), expression of cyclin D1 and cyclin A were low. Scale bar, 20mm.

Article Snippet: Cyclin D1 siRNA (si-cyclin D1) strand A: 50-UCGUCGCCACCUGGAUGCU-30, strand B: 50-AGUGGAACCUGGCCGCAAU-30, and strand C: 50-AACAG AUCAUCCGCAAACA-30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncology reports

Article Title: Klotho inhibits the capacity of cell migration and invasion in cervical cancer.

doi: 10.3892/or.2012.1865

Figure Lengend Snippet: Figure 4. Suppression of Wnt/β-catenin pathway by ectopic expression of Klotho. (A) RT-PCR analysis for direct Wnt-target genes such as c-Myc and cyclin D1. (B) Western blot analysis for alteration of GSK-3β, phosphor- GSK-3β, β-catenin expression level by overexpressed Klotho. Lane 1 represents vector-transfected SiHa and lane 2 is for Klotho-transfection. β-actin and γ-tubulin were used as a loading control for RT-PCR and western analysis, respectively.

Article Snippet: Modified sKL expression level and influenced proteins was examined using the antibodies: Polyclonal anti-sKL (T-19, Santa Cruz Biotechnology), cyclin D1 (M-20, Santa Cruz Biotechnology), total β-catenin (6B3, Cell Signaling), active β-catenin (no. 9582, Cell Signaling), GSK3β (no. 2199, Epitomics), phospho-GSK3β (no. 2435, Epitomics), E-cadherin (610181, BD Biosciences), N-cadherin (610920, BD), MMP7 (J-22, Santa Cruz Biotechnology), and MMP9 (no. 3852, Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Control