Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 1. miR-29c regulates the expression of cyclin E at the posttranscriptional level by targeting 3# UTR of cyclin E mRNA in ESCC cells. (A) The public miRNA database (microRNA Targets Version 5) predicted that cyclin E might be a target for miR-29c, and the 3# UTR of human cyclin E mRNA contains a highly conserved binding site from Position 470 to 492 for miR-29c. (B) The full-length 3# UTR of cyclin E complementary DNA containing miR-29c-binding site was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Wt plasmid (Wt). The full-length 3# UTR of cyclin E complementary DNA (cDNA) deleted 22 nt miR-29c-binding sequence was cloned directly downstream of the firefly luciferase gene to create the pMIR-CCNE-Mut plasmid (Mut). (C) KYSE150 cells and EC9706 cells were transfected with 800 ng Wt or Mut reporter plasmid and the increasing doses of Pre-miR-29c or Pre-Scramble (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). After transfected for 24 h, luciferase activity was measured by a dual-luciferase reporter assay. The result was expressed as relative luciferase activity (firefly LUC/renilla LUC). Columns, mean for three experiments; bars, SE. (D) EC9706 cells and KYSE150 cells were transfected with either 30 nmol/l Pre-miR-29c or Pre-Scramble for 48 h. Cyclin E protein levels were measured by western blotting and cyclin E mRNA levels were measured by reverse transcription–PCR.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Sequencing, Transfection, Activity Assay, Reporter Assay, Western Blot, Reverse Transcription

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 2. Inverse correlation between miR-29c expression and cyclin E protein in ESCC cell lines. (A) miR-29c expression in KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells was analyzed by quantitative real-time PCR. The results were presented as relative miR-29c expression, RNU6B served as internal control. The relative value of miR-29c expression of KYSE450 is set at 1. (B and C) The cell lysates of KYSE150, KYSE410, KYSE450, KYSE510 and EC9706 cells were prepared and analyzed by western blotting. The density of each protein band was quantified using LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. The relative value of cyclin E expression in KYSE450 is set at 1. (D) KYSE150 cells were transfected with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l). Forty-eight hours after transfection, miR-29c level was detected by using quantitative real-time PCR. (E and F) The expression of cyclin E was measured by western blotting, after transfecting KYSE150 cells with the increasing doses of Pre-miR-29c (0 nmol/l, 10 nmol/l, 20 nmol/l and 30 nmol/l) for 48 h. The density of each protein band was quantified by LANE 1D Analyzer V4.0 software (Beijing Sage Creation) and b-actin served as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

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

Journal: Carcinogenesis

Article Title: miR-29c induces cell cycle arrest in esophageal squamous cell carcinoma by modulating cyclin E expression.

doi: 10.1093/carcin/bgr078

Figure Lengend Snippet: Fig. 3. miR-29c induced G1/S cell cycle arrest by suppression of cyclin E expression. (A) EC9706 and KYSE150 cells were transfected with 30 nmol/ l Pre-miR-29c, Pre-Scramble or only Lipofectmine 2000 (Mock). Forty-eight hours after transfection was treated with 100 ng/ml nocodazole for 20 h, cells were collected for cell cycle analysis by propidium iodide staining and flow cytometer analysis. The percentage value of G1 fraction between Pre-miR-29c transfected cells and Pre-Scramble or Mock transfected cells was analyzed. P , 0.01. (B) EC9706 cells were transfected with 30 nmol/l Pre-miR-29c along with the expression plasmid pEF-cyclin E, which contains cyclin E open reading frame without 3# UTR. Forty-eight hours after transfection, cells were treated with 100 ng/ml nocodazole for 20 h. The percentage of cells in G1/G0 was determined by flow cytometer. (C) EC9706 cells and KYSE150 cells were transfected with 30 nmol/l Pre-miR-29c, Pre-Scramble or Mock for 48 h. The cells were collected for western blotting using antibody against cyclin D1, cyclin D2, CDK2 and CDK6. b-Actin was used as loading control. Columns, mean for three experiments; bars, SE.

Article Snippet: The primary antibodies used were as follows: cyclin E antibody (mouse monoclonal, 1:1000; Cell Signaling Technology, Beverly, MA), cyclin D1 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), cyclin D2 Antibody (rabbit polyclonal, 1:800; Proteintech Group Inc., Chicago, IL), CDK2 Antibody (rabbit polyclonal, 1:1000; Santa Cruz Biotechnology Inc.), CDK6 Antibody (mouse monoclonal, 1:1000; Santa Cruz Biotechnology Inc.), b-actin (mouse monoclonal, 1:5000; Proteintech Group Inc.).

Techniques: Expressing, Transfection, Cell Cycle Assay, Staining, Cytometry, Plasmid Preparation, Western Blot, Control

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: Oncogene

Article Title: Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

doi: 10.1038/onc.2013.252

Figure Lengend Snippet: Figure 3. HNRNPM colocalizes with IMP-3 in the nucleus and co-regulates the expression of CCND1, D3 and G1. (a) WB analysis of expression of HNRNPM, IMPs, CCND1, D3 and G1 in RD cells transfected with siRNAs anti-HNRNPM. (b) Immunofluorescent staining of RD cells showing the localization of HNRNPM and IMP proteins, confocal microscopy. (c) WB analysis of IMP and HNRNPM proteins in cytoplasmic (cyt) and nuclear (nucl) fractions of RD cells. Cytoplasmic marker: paxillin. (d) Subcellular localization of IMP-3 in RD cells depleted of HNRNPM by two different siRNAs, confocal microscopy. (e) Subcellular localization of IMP-3 in RD cells depleted of HNRNPM by two different siRNAs and treated for 7 h with 25 mM MG132, confocal microscopy. (f) WB showing the expression of IMP-3, CCND1, CCND3 and HNRNPM in RD cells depleted of HNRNPM by two different siRNAs and treated for 7 h with 25 mM MG132. Shown is a typical experiment out of three. (g) Quantification of three experiments described in f.

Article Snippet: IMP 1 (E-20) sc-21026, IMP-3 (N-19) sc-47893, HNRNPM3/4 (2A6) 1000 2000 0 1500 500 72h post-transfection 48h post-transfection 96h post-transfection 24h post-transfection cell death HeyA8 WI38 HFS 1000 2000 0 3000 1000 2000 0 3000 CT R- 1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2siRNA: CT R- 1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2 CT R1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2siRNA: siRNA: N uc le i p er w el l N uc le i p er w el l N uc le i p er w el l Oncogene (2013), 1 – 10 & 2013 Macmillan Publishers Limited sc-20001, CCND1 (DCS-6) sc-20044, CCND3 (D-7) sc-6283, CCNG1 (H-46) sc7865 for WB and CCNG1 (F-5) sc-8016 for IF and paxilin (H-114) sc-5574 were obtained from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Staining, Confocal Microscopy, Marker

Journal: Oncogene

Article Title: Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

doi: 10.1038/onc.2013.252

Figure Lengend Snippet: Figure 5. IMP-3 nuclear localization correlates with HNRNPM expression and with IMP-3 capacity to regulate the CCND1, D3, and G1 expression in human cells. (a) Immunofluorescent staining of indicated human cell lines showing the localization of IMP proteins and HNRNPM; confocal microscopy. (b) Expression levels of IMP-3, HNRNPM, CCND1 and CCND3 in indicated cell lines. (c) Expression levels of CCND1 and D3 proteins in the indicated cell lines transfected with control or IMP-3 siRNA and collected 48 h later (WB). (d) Cells were transfected with control and anti-IMP1-3 siRNAs, live cells were labeled as described in Materials and methods section, and counted in real- time by automated fluorescence microscopy. Cell death values under 5% of total population are not shown.

Article Snippet: IMP 1 (E-20) sc-21026, IMP-3 (N-19) sc-47893, HNRNPM3/4 (2A6) 1000 2000 0 1500 500 72h post-transfection 48h post-transfection 96h post-transfection 24h post-transfection cell death HeyA8 WI38 HFS 1000 2000 0 3000 1000 2000 0 3000 CT R- 1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2siRNA: CT R- 1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2 CT R1 IM P- 1- 1 IM P- 1- 2 IM P- 2- 1 IM P- 2- 2 IM P- 3- 1 IM P- 3- 2siRNA: siRNA: N uc le i p er w el l N uc le i p er w el l N uc le i p er w el l Oncogene (2013), 1 – 10 & 2013 Macmillan Publishers Limited sc-20001, CCND1 (DCS-6) sc-20044, CCND3 (D-7) sc-6283, CCNG1 (H-46) sc7865 for WB and CCNG1 (F-5) sc-8016 for IF and paxilin (H-114) sc-5574 were obtained from Santa Cruz (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Confocal Microscopy, Transfection, Control, Labeling, Microscopy