Journal: Journal of Integrative Agriculture

Article Title: Critical role of cytochrome c1 and its cleavage in porcine reproductive and respiratory syndrome virus nonstructural protein 4-induced cell apoptosis via interaction with nsp4

doi: 10.1016/s2095-3119(17)61670-8

Figure Lengend Snippet: Fig. 1 Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 4 (nsp4) interacts with cytochrome c1 (cyto.c1). A, yeast two hybrid assay. The plasmids pGBKT7-nsp4 and pGADT7-cyto.c1 were co-transformed into the yeast strain Y2HGold and selected on QDO/X/ABA plates. The top and second panels are the respective positive and negative controls. B, HEK 293T cells were transfected to express Flag-cyto.c1, or HA-nsp4 or together. At 24 h post-transfection, the cells were either subjected to direct Western blot analysis or lysed and immunoprecipitated with anti-HA antibodies. The proteins bound to sepharose beads were separated by SDS-PAGE, transferred to PVDF membranes, and probed with the antibodies to Flag and HA. C, the same as B except that nsp4 truncation mutants were used. D, MARC-145 cells were transduced with letiviruses that were expressing GFP or nsp4-GFP in the presence of 8 µg mL–1 polybrene, respectively. The cells were harvested 48 h post-transduction and cell lysates were immunoprecipitated using anti-GFP beads. The proteins bound to sepharose beads were subjected to Western blot analysis using an anti-cytochrome c1 polyclonal antibody or anti-GFP mAb. E, PRRSV nsp4 interacts with cyto.c1 in mammalian cells. Vero cells grown on coverslips in six-well plates were transfected when 60–70% to express the indicated proteins, either alone or pairwise. At 18–24 h post-transfection, the cells were fixed, permeablized, and stained with antibodies to HA and Flag, and examined by confocal microscopy. For double transfections, the merged images are shown at the right.

Article Snippet: Mouse anti-green fluorescent protein (GFP) mAb (66002-1-Ig) and rabbit anti-cytochrome c1 polyclonal antibodies (10242-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Virus, Y2H Assay, Transformation Assay, Transfection, Western Blot, Immunoprecipitation, SDS Page, Transduction, Expressing, Staining, Confocal Microscopy

Journal: Oral Science International

Article Title: Down‐regulated expression of family with sequence similarity 3, member B (FAM3B), in oral squamous cell carcinoma

doi: 10.1016/s1348-8643(12)00004-3

Figure Lengend Snippet: Fig. 1. Family with sequence similarity 3, member B (FAM3B) mRNA expression in oral squamous cell carcinoma (OSCC)-derived cell lines. FAM3B mRNA levels were

Article Snippet: Immunohistochemical analysis Immunohistochemical analysis was performed on 4- m sections of paraffin-embedded specimens using anti-FAM3B polyclonal antibody (Proteintech).

Techniques: Sequencing, Expressing, Derivative Assay

Journal: Oral Science International

Article Title: Down‐regulated expression of family with sequence similarity 3, member B (FAM3B), in oral squamous cell carcinoma

doi: 10.1016/s1348-8643(12)00004-3

Figure Lengend Snippet: Fig. 2. Family with sequence similarity 3, member B (FAM3B) protein expression in oral

Article Snippet: Immunohistochemical analysis Immunohistochemical analysis was performed on 4- m sections of paraffin-embedded specimens using anti-FAM3B polyclonal antibody (Proteintech).

Techniques: Sequencing, Expressing

Journal: Oral Science International

Article Title: Down‐regulated expression of family with sequence similarity 3, member B (FAM3B), in oral squamous cell carcinoma

doi: 10.1016/s1348-8643(12)00004-3

Figure Lengend Snippet: Fig. 3. Expression profile of family with sequence similarity 3, member B (FAM3B) mRNA

Article Snippet: Immunohistochemical analysis Immunohistochemical analysis was performed on 4- m sections of paraffin-embedded specimens using anti-FAM3B polyclonal antibody (Proteintech).

Techniques: Expressing, Sequencing

Journal: Oral Science International

Article Title: Down‐regulated expression of family with sequence similarity 3, member B (FAM3B), in oral squamous cell carcinoma

doi: 10.1016/s1348-8643(12)00004-3

Figure Lengend Snippet: Fig. 4. Immunohistochemical (IHC) analysis of family with sequence similarity 3, member B (FAM3B) protein expression in primary oral squamous cell carcinoma (OSCC) samples. Representative results of IHC analysis for FAM3B in normal oral tissue (A, B) and primary OSCC (C, D). Original magnification: ×100 (A, C) and ×400 (B, D). Positive immunoreactions

Article Snippet: Immunohistochemical analysis Immunohistochemical analysis was performed on 4- m sections of paraffin-embedded specimens using anti-FAM3B polyclonal antibody (Proteintech).

Techniques: Immunohistochemical staining, Sequencing, Expressing

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: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 1 CstF1 interacts with both BARD1 and p53 to form a protein complex. (a) Diagram of BARD1, CstF1 and p53 derivatives. (b) Requirement of p53 C-terminal domain for both CstF1 and BARD1 interaction. Recombinant His-p53 or the indicated His-p53 derivatives were incubated with purified GST, GST-CstF1 or GST-BARD1. Protein samples were treated with RNase A. Bound proteins were eluted and analyzed by western blot with anti-p53 antibodies. Five percent of His-p53 or His-p53 derivatives used in the reaction are shown as input. (c) CstF1, BARD1 and p53 can coexist in complexes. Either GST-BARD1 or GST-CstF1 was immobilized in glutathione-Sepharose beads to test its ability to bind CstF1/BARD1 and p53 from NE of HeLa cells, while increasing concentrations of recombinant His-p53 (5, 10 and 20 ng) were added. Bound proteins were detected by immunoblotting with CstF1 and p53 antibodies. The pellets (PD) and the supernatant (SN) were analyzed. (d) p53, CstF and BARD1 co-immunoprecipitate from NEs of MCF7 cells treated with UV irradiation. CstF and p53 co-immunoprecipitation is irrespective of UV irradiation. Co-immunoprecipitation assays were performed using NEs of cells exposed or not to UV irradiation and allowed to recover for 2 h as described before. NEs were immunoprecipitated with either anti-CstF2, to ensure that any detected interactions were between p53 and intact CstF, or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Recombinant, Incubation, Western Blot, Irradiation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Control

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 4 The S241F mutation in p53 disrupts the BARD1, CstF and p53 interaction and decreases the inhibitory effect of p53 on 30 cleavage, and all this is restored by WT-p53 expression. (a) CstF, p53 and BARD1 do not co-immunoprecipitate from NE of DLD-1 cells that express S241F mutant p53. NEs were immunoprecipitated with either anti-CstF2 or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, BARD1, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input. (b) CstF, BARD1 and p53 co-immunoprecipitate from NEs of D-A2 cells following induction of WT-p53 expression. D-A2 cells were grown in the absence of Dox to induce the expression of WT-p53. Samples were analyzed as in (a). (c) The induced expression of WT-p53 in D-A2 cells inhibits pre-mRNA 30 cleavage. NEs from DLD-1 (left panel) and D-A2 cells (right panel) non-treated or treated with Dox and/or UV irradiation, and allowed to recover for the times indicated in the figure, were analyzed for L3 pre-mRNA 30 cleavage. Positions of pre-mRNA and the 50 cleavage product are indicated. (d) The S241F mutation in p53 abolishes the inhibition of 30 cleavage. NEs from HeLa cells were preincubated with no addition or increasing amounts of recombinant His-p53 or His-p53 (S241F) derivative (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Irradiation, Inhibition, Recombinant, Incubation

Journal: Obesity (Silver Spring, Md.)

Article Title: Characterization of herpes virus entry mediator as a factor linked to obesity.

doi: 10.1038/oby.2009.250

Figure Lengend Snippet: Figure 1 Data from real-time reverse transcription–PCR showing herpes virus entry mediator (HVEM) gene expression in (a) subcutaneous and visceral human adipose tissue and (b) subcutaneous adipose tissue of lean vs. obese subjects. (c) Western blot analysis of HVEM in subcutaneous human adipose tissue in lean and obese subjects. Relative mRNA levels were normalized to the levels of cyclophilin A and densitometric analyses of HVEM protein were normalized to β-actin. Western blot bands are representative of n = 12 subjects. *P < 0.05.

Article Snippet: Membranes were immunoblotted with antihuman HVEM (Proteintech, Chicago, IL) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Reverse Transcription, Virus, Gene Expression, Western Blot

Journal: Obesity (Silver Spring, Md.)

Article Title: Characterization of herpes virus entry mediator as a factor linked to obesity.

doi: 10.1038/oby.2009.250

Figure Lengend Snippet: Figure 2 Herpes virus entry mediator (HVEM) gene expression in adipocytes. (a) Data from real-time reverse transcription–PCR (RT-PCR) showing HVEM gene expression in stromovascular cells (SVCs) and mature adipocytes (ad) separated from human adipose tissue. Data are means ± s.e.m. (n = 8). (b) Data from real-time RT-PCR and (c) western blot analysis of HVEM in primary culture of human preadipocytes during the differentiation process. Relative mRNA levels were normalized to the levels of cyclophilin A and densitometric analyses of HVEM protein were normalized to β-actin. Data are representative of three different experiments. *P < 0.05.

Article Snippet: Membranes were immunoblotted with antihuman HVEM (Proteintech, Chicago, IL) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Virus, Gene Expression, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot