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: 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: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 1. Pivotal role of Glu246 and Asp247 in the conformation of the RRM2 domain of TDP-43. A. a, Scheme showing the alignment of RRM2 subdomains (residues 232–270 in human TDP-43) in multiple species. Glu246 (E246) and Asp247 (D247) are preserved across all species. b, Chemical properties of amino acid substitution mutants of E246 or D247. E246Q and D247N were used to create substitution mutants with minimal alteration of side chains. E246G and D247G were designed so that the effects of the side chains were eliminated, and the flexibility of the amide bonds was increased. B. Western blot analysis of recombinant RRM2 proteins of wild-type (WT), E246Q/D247N (QN), and E246G/D247G (GG), using an anti-TDP-43 rabbit polyclonal antibody that recognizes the RRM2 domain (Proteintech). The E246G/D247G mutant RRM2 showed marked oligomerization even when incubated at 4uC (vertical line). Note that considerable RRM2 dimers or oligomers were dissociated into monomers in the presence of DTT. Arrowhead and double arrowheads indicate RRM2 monomer and dimers, respectively. Asterisk possibly indicates heat-related high molecular complexes comprising RRM2 domain. C. Thioflavin T (ThT) fluorescence assay showing amyloid fibril formation of RRM2 with mutations in E246 and D247. Each value indicates averaged RFU of ThT with standard error of mean from triplicates. *p,0.01 vs. RRM2 WT at RT by one-way ANOVA with Newman-Keuls test. D. Superdex 75 size exclusion chromatography of recombinant RRM2 domain of WT (a), E246Q/D247N (QN, b), and E246G/D247G (GG, c). a, WT RRM2 alone was exclusively monomeric in its native condition (unfilled circle). Heat denaturation at 70uC for 10 min induced higher molecular assembly (filled circle). Arrowhead indicates the RRM2 oligomer. b, E246Q/D247N (QN) mutant RRM2 without stress was predominantly monomeric (unfilled circle). Heat denaturation markedly increased the ratio of oligomers to monomers (filled circle). c, E246G/D247G (GG) mutant existed as a mixture of monomer and oligomers at the baseline condition (unfilled circle). Higher molecular species were prominently

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Western Blot, Recombinant, Mutagenesis, Incubation, Fluorescence, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 2. D247 predominantly governs the conformation of RRM2. A. Coomassie staining of recombinant RRM2 protein with either single or double mutation(s) at E246 and D247 incubated for 24 hr at 4uC by denaturing SDS-PAGE. The effects of oligomerization were greater with the single mutant at D247 than at E246. Oligomerization is most prominent in the double E246G/D247G mutant. B. Size exclusion chromatography of RRM2 proteins of E246G and D247G incubated at 4uC (a) and 70uC for 10 min with 24 hr post incubation at 4uC. (b). The effect of oligomerization is more pronounced in the D247G mutant RRM2 than in the E246G in both the 4uC and 70uC conditions. Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). doi:10.1371/journal.pone.0052776.g002

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Staining, Recombinant, Mutagenesis, Incubation, SDS Page, Size-exclusion Chromatography

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 3. Substitution mutants of full-length TDP-43 at E246 and D247 are readily misfolded. A. Confocal micrographs of HEK293A cells overexpressing EGFP-fused full-length TDP-43 (a, b, wild-type (WT), c, d, E246G, e, f, D247G, g, h, E246G/D247G). i, j, Percentages of transfected HEK293A cells harboring multiple puncta or inclusions (i, arrowheads) or displaying nucleus-excluded TDP-43 (unfilled arrowhead). (j). Scale bar indicates 30 mm. Data were expressed as the mean 6 SEM (N = 7–10). *p,0.05 vs. WT, #p,0.05 vs. E246G by one-way ANOVA with Newman-Keuls test. NS indicates not significant vs. WT. B. a, Western blotting showing the increased detergent-insolubility of TDP-43 with mutations at E246/D247, with defective nucleus localizing signal (mNLS), or devoid of RRM2 domain (DRRM2). Lysates from HEK293A cells transiently transfected with TDP-43- EGFP were separated into 1% TritonX100-soluble or -insoluble fractions. Top panel, anti-GFP; middle panel, anti-actin; bottom panel, anti-GAPDH. The GAPDH blot validates the successful separation between detergent-soluble and -insoluble components. b, Quantified insolubility of TDP-43-EGFP proteins with or without mutation at E246/D247 to glycine. Relative TDP-43-EGFP in the detergent-soluble or -insoluble fraction was obtained from the ratio of the GFP density to actin density from the densitometric value in each fraction (designated as insoluble TDP or soluble TDP, respectively). Insolubility index was obtained from the ratio of insoluble TDP to soluble TDP, and each value was standardized by the average ratio of WT. Data were expressed as the mean 6 SEM of four experiments.*p,0.05 vs. WT TDP-43-EGFP by one-way ANOVA with Newman-Keuls test. C. Size exclusion chromatography and Western blotting indicating the existence of oligomeric and monomeric states of full-length TDP-43 in cells. WT and E246G/ D247G (GG) TDP-43-FLAG genes were expressed in HEK293A cells. Cells were sonicated in PBS, and the supernatants were fractionated by a Superose 12 column (10/300) at a flow rate of 0.5 mL/min in PBS. Fractionated cell extracts were applied to Western blotting by anti-TDP-43 (Proteintech). Mutant TDP-43 (GG) proteins were collected in a larger fraction than 88–440 kDa. The molecular size markers thyroglobulin (669 kDa), ferritin (440 kDa), Mn-SOD (88 kDa), ovalbumin (43 kDa), and RNase (13.7 kDa) were eluted under the same conditions. Abs280 is presented to show the equal amount of proteins between WT and the GG mutant in each fraction. doi:10.1371/journal.pone.0052776.g003

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Western Blot, Mutagenesis, Size-exclusion Chromatography, Sonication

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 4. Oligomerization affects the nucleotide interaction and RNA splicing efficiency of TDP-43. A. Exon 9 skipping assay showing that mutations at E246 and D247 affect the RNA splicing activity of full-length TDP-43. a, Agarose gel electrophoresis of PCR products (top). Western blot analysis of the total cell lysates using anti-EGFP (middle) and -actin (bottom) antibodies was also shown. b, Quantification of spliced and unspliced fragments using densitometry. Each value is the ratio of spliced to unspliced PCR products. Data is mean 6 standard error of mean from triplicates. *p,0.01 vs. Wild-type (WT) TDP-43 by one-way ANOVA with Newman-Keuls test. B. Size exclusion chromatography for recombinant RRM2 proteins of WT (a), or mutants with E246Q/D247N (QN, b) or E246G/D247G (GG, c), and for (TG)12 oligonucleotides. Mixtures of RRM2 mutants and (TG)12 oligonucleotides were centrifuged at 15,0006g for 20 min and subjected to a Superdex75 (10/300) column at a flow rate of 0.5 mL/min in PBS. Only the RRM2 monomer showed a molecular shift with the (TG)12 oligonucleotides to a single peak, indicating their association (arrowheads). Molecular size markers are as follows: bovine serum albumin (66 kDa), ovalbumin (43 kDa), superoxide dismutase 1 (32 kDa), myoglobin (17.6 kDa), and aprotinin (6.5 kDa). *1 indicates free monomeric RRM2, *2 indicates oligomeric RRM2. Note that there is no peak for free (TG)12, indicating all the (TG)12 was bound to RRM2 monomers. doi:10.1371/journal.pone.0052776.g004

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Activity Assay, Agarose Gel Electrophoresis, Western Blot, Size-exclusion Chromatography, Recombinant

Journal: PloS one

Article Title: Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43.

doi: 10.1371/journal.pone.0052776

Figure Lengend Snippet: Figure 5. 3B12A recognizes cytosol-redistributed TDP-43. A–D, SHSY-5Y cells were transiently transfected with TDP-43-EGFP of wild type (WT), mutants with defective NLS (mNLS), or deletion mutant of RRM2 deletion (DRRM2) (EGFP shown as green). At 48 h after transfection, cells were fixed and stained with 3B12A (red). DAPI was used for counterstaining (blue). A. a–f, Transfected or endogenous WT TDP-43 was rarely stained by 3B12A (unfilled arrowheads). Occasionally, cells with very high fluorescence were labeled (arrowhead). B. Cytosolic redistributed TDP-43 (mNLS) was preferentially stained by 3B12A (arrowheads). 3B12A recognized the mNLS mutant of TDP-43-EGFP even at moderate expression levels, regardless of aggregate formation. c–e are high power fields of a–b. C. Nuclear-excluded WT TDP-43 is recognized by 3B12A. WT TDP-43-EGFP expressing SHSY-5Y cells exposed to 5 mM lactacystin were fixed and stained with 3B12A (arrowheads). D. No reactivity of 3B12A to TDP-43-EGFP devoid of RRM2 (DRRM2) (unfilled arrowheads). E. Immunoprecipitation experiment showing that 3B12A preferentially recognized NLS-defective TDP-43 in cell lysates. HEK293A cells were transiently transfected with WT, mNLS, or FALS mutant (A315T and Q331K) forms of TDP-43-FLAG. Total lysates were immunoprecipitated with the 3B12A. Western blot analysis using a rabbit polyclonal anti-FLAG antibody showed that 3B12A predominantly recognized the defective NLS, but more weakly recognized the WT and FALS-linked mutant forms of TDP-43. doi:10.1371/journal.pone.0052776.g005

Article Snippet: To detect recombinant RRM2 proteins or full-length TDP-43 in cultured cells, rabbit polyclonal anti-TDP-43 antibody (ProteinTech Group Inc. Chicago, IL) was used (1:1000).

Techniques: Transfection, Mutagenesis, Staining, Fluorescence, Labeling, Expressing, Immunoprecipitation, Western Blot

Journal: Biochimica et biophysica acta

Article Title: RNA helicase A is not required for RISC activity.

doi: 10.1016/j.bbagrm.2013.07.008

Figure Lengend Snippet: Fig. 1. RHA protein is not required for siRNA activity. A) Western analyses of RHA protein in cells treated with 5 nM RHA siRNA (+siRNA) or 50 nM RHA ASO (+ASO). RPL4 was detected and served as a loading control. UTC, cells transfected without siRNA or ASO. B) qRT-PCR analysis for the levels of PTEN mRNA. HeLa cells pre-treated with RHA siRNA (RHA-siRNA) or ASO (RHA-ASO) as used in panel A were transfected for 4 h with PTEN siRNA at different concentrations, as indicated. Total RNA was prepared and PTEN mRNA levels were detected using qRT-PCR. C) qRT-PCR analyses for the levels of NPM1 mRNA in RHA reduced cells transfected with different concentrations of NPM1 siRNAs, as in panel B. D) qRT-PCR for the level of RHA mRNA in cells transfected with 5 nM RHA-si-544 or 50 nM RHA-ASO. E) qRT-PCR assay for the level of PTEN mRNA in cells pre-transfected for 24 h with RHA-si-544 or RHA-ASO as in panel D, followed by transfection with different concentrations of PTEN-siRNA, as in panel B. F) qRT-PCR for PTEN mRNA in cells pre-treated with 5 nM luciferase siRNA (Leu-si) or RHA-si-544. The activity of sub- sequently transfected PTEN-siRNA was determined as in panel E. The error bars represent standard deviation from three independent experiments in all panels.

Article Snippet: RPL4 antibody (11302-1-AP, 1:1000) was from Proteintech.

Techniques: Activity Assay, Western Blot, Control, Transfection, Quantitative RT-PCR, Luciferase, Standard Deviation

Journal: Biochimica et biophysica acta

Article Title: RNA helicase A is not required for RISC activity.

doi: 10.1016/j.bbagrm.2013.07.008

Figure Lengend Snippet: Fig. 2. Pre-treatment with NCL1-siRNA, but not ASO, also reduced RISC activity. A) Western analysis for NCL1 protein levels in cells transfected with 5 nM NCL1 siRNA or 50 nM ASO, as in Fig. 1A. RPL4 was detected and served as a loading control. B) qRT-PCR analysis for the levels of PTEN mRNA in control or different NCL1 reduced cells that were transfected again with PTEN siRNA, as in panel B. C) qRT-PCR for the levels of NPM1 mRNA in control or NCL1 reduced cells that were transfected with NPM1 siRNA at different concentrations. The error bars represent standard deviation from three parallel experiments in all panels.

Article Snippet: RPL4 antibody (11302-1-AP, 1:1000) was from Proteintech.

Techniques: Activity Assay, Western Blot, Transfection, Control, Quantitative RT-PCR, Standard Deviation

Journal: Biochimica et biophysica acta

Article Title: RNA helicase A is not required for RISC activity.

doi: 10.1016/j.bbagrm.2013.07.008

Figure Lengend Snippet: Fig. 4. siRNA loading can be affected by the pre-transfected siRNAs in a dose dependent manner. A) Western analysis for RHA protein in cells transfected with different concentrations of siRNAs. RPL4 was detected and served as a loading control. B) qRT-PCR for the cellular levels of transfected PTEN siRNA in cells pre-transfected with 0.5 or 5.0 nM RHA siRNAs. C) Less PTEN siRNA was associated with Ago2 in cells pre-transfected with higher concentration of RHA siRNA. Immunoprecipitation with Ago2 antibody and qRT-PCR for PTEN and RHA siRNAs were performed as in Fig. 3D. The relative levels of RHA siRNA were calculated based on the level of RHA siRNA co-precipitated with Ago2 from cells transfected with 5 nM RHA siRNA. D) Western analysis for NCL1 protein in cells treated with NCL1 siRNAs. RPL4 served as a loading control. E) Total cellular levels of transfected PTEN siRNA in different test cells, as in panel B. F) The relative levels of Ago2-associated PTEN and NCL1 siRNAs in different test cells, as in panel C. The relative levels of NCL1 siRNA were calculated based on the level of NCL1 siRNA co-precipitated with Ago2 from cells transfected with 5 nM NCL1 siRNA. The error bars represent standard deviation from three parallel experiments in all panels.

Article Snippet: RPL4 antibody (11302-1-AP, 1:1000) was from Proteintech.

Techniques: Transfection, Western Blot, Control, Quantitative RT-PCR, Concentration Assay, Immunoprecipitation, Standard Deviation

Journal: Biochimica et biophysica acta

Article Title: RNA helicase A is not required for RISC activity.

doi: 10.1016/j.bbagrm.2013.07.008

Figure Lengend Snippet: Fig. 6. ASO-mediated reduction of MOV10 impaired siRNA activity. A) Western analysis for the levels of MOV10 protein in cells transfected with 0.75 or 6.0 nM MOV10 siRNA or 50 nM ASO. RPL4 was detected and served as a loading control. B) qRT-PCR for the levels of PTEN mRNA in control cells or different MOV10-depleted cells that were subsequently transfected again with PTEN siRNA at different concentrations. C) PTEN siRNA association with Ago2 was not affected by MOV10 reduction. The levels of transfected cellular or Ago2-co-precipitated PTEN siRNA were determined using qRT-PCR. D) RT-PCR for the levels of MOV10 mRNA in cells treated for 24 h with different concentrations of MOV10-ASOs. E) Western analysis for MOV10 protein. GAPDH was detected and served as a loading control. F) qRT-PCR for the levels of PTEN mRNA in different ASO treated cells that were transfected again with different concentrations of PTEN siRNA for 4 h, as described in Materials and methods. The error bars represent standard deviation from three parallel experiments in all panels.

Article Snippet: RPL4 antibody (11302-1-AP, 1:1000) was from Proteintech.

Techniques: Activity Assay, Western Blot, Transfection, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

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