Journal: Cancer biology & therapy

Article Title: A novel peroxisome proliferator-activated receptor delta antagonist, SR13904, has anti-proliferative activity in human cancer cells.

doi: 10.4161/cbt.8.13.8691

Figure Lengend Snippet: Figure 4. SR13904 exerts inhibitory effects on the cell cycle. (A) A representative experiment in which A549 cells were grown in phenol-red-free DMEM + 2% charcoal-treated FBS ± SR13904 (20 μM). Cells were treated in the exponential phase and assessed by flow cytometry at 24 and 48 h. (B) Statistical analysis of cell cycle distribution (G1, S and G2 phases) from three experiments. All differences (SR13904-treated vs. vehicle control-treated) were highly significant. p values for the G1 phase are shown. **p = 0.001–0.01, ***p = <0.001 (C) Western analysis of select cell cycle proteins. A549 cells were grown in phenol-red-free DMEM + 0.5% charcoal-treated FBS and exposed to the ligand. Relative protein levels were assessed at 24 h and 48 h following addition of SR13904 (20 μM) or control. (D) mRNA analysis of CDK2, CDK4 and cyclin D1. A549 cells were grown and treated as in (C). Real-time PCR for CDK2, CDK4 and cyclin D1 were performed as described in Materials and Methods. Each experimental measurement was normalized to the corresponding GAPDH mRNA levels. For CDK2 (24 and 48 h time points) and cyclin D1 (48 h) time point, the differences (SR13904- treated vs. vehicle control-treated) were significant. *p = 0.01–0.05, **p = 0.001–0.01.

Article Snippet: CDK4 (#2906), cyclin D1 (#2922), cyclin E (#4129), p21 (#2947) and p27 (#2552) antibodies were purchased from Cell Signaling Technology.

Techniques: Flow Cytometry, Control, Western Blot, Real-time Polymerase Chain Reaction

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: Impact of Mucin1 knockdown on the phenotypic characteristics of the human hepatocellular carcinoma cell line SMMC-7721.

doi: 10.3892/or.2014.3136

Figure Lengend Snippet: Figure 3. Knockdown of MUC1 expression alters the β‑catenin signaling pathway by blocking β‑catenin translocation to the nucleus. (A) Cell lysates from NC and MR1-D4 clones were subjected to immunoprecipitation (IP) with anti-MUC1-CT antibody or normal IgG and then immunoblotted (IB) with anti‑β‑catenin antibody. Whole cell lysate (WCL) was not subjected to immunoprecipitation. (B) Cytoplasmic extracts from SMMC-7721, NC, MR1-C6 and MR1-D4 cells were analyzed by western blotting for the expression of β‑catenin. Cytoplasmic IκBα was used as a protein loading control. (C) Nuclear extracts from SMMC‑7721, NC, MR1-C6 and MR1-D4 cells were detected by western blotting to assess the levels of nuclear β‑catenin. Lamin B1 served as the nuclear loading control. (D) NC, MR1-C6 and MR1-D4 cells were transiently transfected with TOPflash and FOPflash plasmids. Relative luciferase activity was cal culated as the ratio of TOPflash/FOPflash luciferase activity, and each value was normalized to the luciferase activity of the internal control pRL-TK reporter plasmid. (E) mRNA levels of cyclin D1 and c-Myc in NC, MR1-C6 and MR1-D4 cells were detected by qRT-PCR and normalized to β‑actin. Bars represent the relative mRNA level when compared to the NC cells. (F) Cell lysates were analyzed by western blotting for the expression of cyclin D1 and c-Myc. β‑actin was used as a loading control. Data are expressed as the means ± SDs of 3 independent experiments. *P<0.05 compared with NC.

Article Snippet: The primary antibodies used were antibodies against MUC1 (GP1.4) (1:2,000; NeoMarkers), c-Myc (1:1,000), cyclin D1 (1:1,000), β-actin (1:2,000), IκBα (1:2,000) and Lamin B1 (1:2,000; all from Epitomics, Burlingame, CA, USA), β-catenin (1:1,000; BD Biosciences), E-cadherin (Proteintech), caspase-3 (Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, Blocking Assay, Translocation Assay, Clone Assay, Immunoprecipitation, Western Blot, Control, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Quantitative RT-PCR

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