Journal: BMC Microbiology

Article Title: Real-time quantitative PCR assay development and application for assessment of agricultural surface water and various fecal matter for prevalence of Aliarcobacter faecis and Aliarcobacter lanthieri

doi: 10.1186/s12866-020-01826-3

Figure Lengend Snippet: List of reference strains of target and other bacterial species and strains used in this study

Article Snippet: 58 , S. enterica subsp. houtenae , – , ATCC 29932.

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: Cleavage of Desmosomal Cadherins Promotes γ-Catenin Degradation and Benefits Wnt Signaling in Coxsackievirus B3-Induced Destruction of Cardiomyocytes

doi: 10.3389/fmicb.2020.00767

Figure Lengend Snippet: γ-Catenin inhibits target gene transcription of Wnt/β-catenin signaling during CVB3 infection. (A,B) HeLa cells were transfected with the γ-catenin plasmid for 36 h to overexpress γ-catenin and then infected with CVB3 at a MOI of 10 or sham-infected. Cellular RNAs were extracted at 6 h pi and then subjected to qPCR analysis of c-myc (A) and MMP9 (B) mRNAs using specific primers. (C,D) HeLa cells were transfected with γ-catenin siRNA for 48 h to silence γ-catenin expression and then infected with CVB3 at a MOI of 10 or sham-infected. Cellular RNAs were extracted at 6 h pi and the mRNA levels of c-myc and MMP9 were measured as described in (A) . The transcriptional levels of target genes were normalized to that of GAPDH and statistically analyzed, n = 3, **** p < 0.0001.

Article Snippet: The γ-catenin siRNA (human) and DSC-2 siRNA (human) were purchased from Santa Cruz (United States).

Techniques: Infection, Transfection, Plasmid Preparation, Expressing

Journal: Frontiers in Microbiology

Article Title: Cleavage of Desmosomal Cadherins Promotes γ-Catenin Degradation and Benefits Wnt Signaling in Coxsackievirus B3-Induced Destruction of Cardiomyocytes

doi: 10.3389/fmicb.2020.00767

Figure Lengend Snippet: Knockdown of γ-catenin expression enhances CVB3 replication. HeLa cells were transfected with γ-catenin siRNA or scrambled siRNA for 48 h and then infected with CVB3 at a MOI of 10 or sham-infected for 4 h. Part of the cell culture was used for qPCR to measure CVB3 2A RNA (A), and the other part was used either for Western blot analysis of CVB3 VP1 protein (B) or for plaque assay to determine the released viral particles (C) . Data were statistically analyzed and graphically presented, n = 3, ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: The γ-catenin siRNA (human) and DSC-2 siRNA (human) were purchased from Santa Cruz (United States).

Techniques: Knockdown, Expressing, Transfection, Infection, Cell Culture, Western Blot, Plaque Assay

Journal: Frontiers in Microbiology

Article Title: Cleavage of Desmosomal Cadherins Promotes γ-Catenin Degradation and Benefits Wnt Signaling in Coxsackievirus B3-Induced Destruction of Cardiomyocytes

doi: 10.3389/fmicb.2020.00767

Figure Lengend Snippet: γ-Catenin supports the induction of interferon β1 and its stimulated genes. HeLa cells were transfected with siRNA to knockdown the expression of γ-catenin and then infected with CVB3 at 10 MOI. Scrambled siRNA was used as a negative control. Cellular RNAs were extracted at 6 h pi and then subjected to qPCR analysis of the mRNA levels of IFNB1 gene (A) , MDA5 (B) , MAVS (C) , and ISG15 (D) . The transcriptional level of each gene was normalized to GAPDH level and statistically analyzed, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001, **** p < 0.0001.

Article Snippet: The γ-catenin siRNA (human) and DSC-2 siRNA (human) were purchased from Santa Cruz (United States).

Techniques: Transfection, Knockdown, Expressing, Infection, Negative Control

Journal: Molecular Biology of the Cell

Article Title: A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A 2 α in endothelial cells

doi: 10.1091/mbc.E11-08-0694

Figure Lengend Snippet: α-Catenin regulates the relocalization of cPLA 2 α to the Golgi apparatus upon cell confluence. (A) HUVECs treated with siRNA against α-catenin, β-catenin, γ-catenin, δ-catenin, or scrambled control siRNA for 48 h were serum starved (4 h) and stimulated with VEGF-A (25 ng/ml, 30 min). Lysates were analyzed by immunoblotting as indicated. Shown are representative blots from four independent experiments. (B) Cells treated with siRNA as in A were analyzed for VE-cadherin protein expression, quantified, and expressed as a percentage of scrambled siRNA-treated levels. Results represent five independent experiments. (C) Cells treated with siRNA as in A were analyzed by immunoblotting for catenin expression. Results quantified from five independent experiments are expressed as a percentage of scrambled siRNA-treated levels. (D) HUVECs treated with siRNA against either α-catenin or a scrambled sequence were fixed and processed by immunostaining with anti-cPLA 2 α, anti-VE-cadherin, and a combination of anti-TGN46/anti–α-catenin antibodies. Scale bar, 20 μm. (E) HUVECs transfected with either scrambled siRNA or siRNA against δ-catenin were grown to confluence, fixed, and immunostained with antibodies against cPLA 2 α (green), VE-cadherin (red), and a combination of TGN46 and δ-catenin (purple). Scale bar, 20 μm. (F) HUVECs treated with siRNA against δ-catenin or scrambled control were left confluent (upper) or scratch wounded and recovered for 18 h (middle, bottom) prior to fixation and analysis by immunostaining as indicated. Wound directions are indicated by white line. (G) HUVECs transfected with indicated siRNA were processed as outlined in D and E. The ratio of cPLA 2 α to TGN46 fluorescence intensity at the Golgi apparatus was determined and expressed as a percentage of scrambled siRNA-treated cells. Quantified results represent >200 cells from at least four independent experiments. *p < 0.05, **p < 0.01.

Article Snippet: HUVECs were transfected with 6 pmol/13-mm 2 dish of nontargeting annealed control siRNA (Scrambled D-001210-01; Dharmacon, Lafayette, CO), annexin A1 (sc-29198, Santa Cruz Biotechnology, or 146987, Ambion, Austin, TX), cPLA 2 α (Ambion; Herbert et al. , 2009 ), α-catenin (sc-29190, Santa Cruz Biotechnology; or L-010505, Dharmacon), β-catenin siRNA (s436; Ambion), γ-catenin (sc-29324, Santa Cruz Biotechnology; or L-011708, Dharmacon), δ-catenin (sc-43021, Santa Cruz Biotechnology; or L-012572, Dharmacon), PAR3 (s32128, s32127, s32126; Ambion) or VE-cadherin siRNA (sc-36814, Santa Cruz Biotechnology; or s2780, Ambion) for 5–6 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

Techniques: Western Blot, Expressing, Sequencing, Immunostaining, Transfection, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A 2 α in endothelial cells

doi: 10.1091/mbc.E11-08-0694

Figure Lengend Snippet: Loss of α-catenin is unable to induce cPLA 2 α redistribution from the Golgi in the absence of δ-catenin. (A) HUVECs treated with either or both α-catenin– and δ-catenin–targeted siRNA or a scrambled control were fixed and processed with the indicated antibodies. (B) Cells treated as in A were scratch wounded, recovered for 18 h, fixed, and processed as described. Scale bar, 20 μm. (C) Ratio of cPLA 2 α:TGN46 localization at the Golgi apparatus was determined from B and expressed relative to ratios from confluent scrambled siRNA-treated cells. *p < 0.05 compared with scrambled siRNA-treated control.

Article Snippet: HUVECs were transfected with 6 pmol/13-mm 2 dish of nontargeting annealed control siRNA (Scrambled D-001210-01; Dharmacon, Lafayette, CO), annexin A1 (sc-29198, Santa Cruz Biotechnology, or 146987, Ambion, Austin, TX), cPLA 2 α (Ambion; Herbert et al. , 2009 ), α-catenin (sc-29190, Santa Cruz Biotechnology; or L-010505, Dharmacon), β-catenin siRNA (s436; Ambion), γ-catenin (sc-29324, Santa Cruz Biotechnology; or L-011708, Dharmacon), δ-catenin (sc-43021, Santa Cruz Biotechnology; or L-012572, Dharmacon), PAR3 (s32128, s32127, s32126; Ambion) or VE-cadherin siRNA (sc-36814, Santa Cruz Biotechnology; or s2780, Ambion) for 5–6 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A 2 α in endothelial cells

doi: 10.1091/mbc.E11-08-0694

Figure Lengend Snippet: Depletion of α-catenin does not affect VE-cadherin plasma membrane levels. (A) Confluent HUVECs lysates were immunoprecipitated with protein G Sepharose and antibodies against either α-catenin, cPLA 2 α, or a rabbit isotype control Ig. Bound complexes were separated by SDS–PAGE and immunoblotted with indicated antibodies. (B) Biotinylated surface proteins from HUVECs treated with siRNA against indicated catenins, VE-cadherin, or a scrambled sequence were isolated with NeutrAvidin agarose and analyzed by immunoblotting with antibodies against indicated proteins. (C) Immunoblots from B were quantified and collated from three independent experiments. **p < 0.01. (D) Whole-cell lysates from A were analyzed by immunoblotting with indicated antibodies.

Article Snippet: HUVECs were transfected with 6 pmol/13-mm 2 dish of nontargeting annealed control siRNA (Scrambled D-001210-01; Dharmacon, Lafayette, CO), annexin A1 (sc-29198, Santa Cruz Biotechnology, or 146987, Ambion, Austin, TX), cPLA 2 α (Ambion; Herbert et al. , 2009 ), α-catenin (sc-29190, Santa Cruz Biotechnology; or L-010505, Dharmacon), β-catenin siRNA (s436; Ambion), γ-catenin (sc-29324, Santa Cruz Biotechnology; or L-011708, Dharmacon), δ-catenin (sc-43021, Santa Cruz Biotechnology; or L-012572, Dharmacon), PAR3 (s32128, s32127, s32126; Ambion) or VE-cadherin siRNA (sc-36814, Santa Cruz Biotechnology; or s2780, Ambion) for 5–6 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

Techniques: Immunoprecipitation, SDS Page, Sequencing, Isolation, Western Blot

Journal: Molecular Biology of the Cell

Article Title: A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A 2 α in endothelial cells

doi: 10.1091/mbc.E11-08-0694

Figure Lengend Snippet: F-Actin is required for tethering cPLA 2 α to the Golgi apparatus in confluent endothelial cells. (A) Confluent HUVECs were treated with CytD (1 μM) for the indicated times prior to fixation and processing with antibodies against cPLA 2 α and VE-cadherin, together with Alexa Fluor 633–phalloidin. Scale bar, 20 μM. (B) Confluent HUVECs were treated for 4 h with CytD (200 nM) prior to fixation and localization analysis. Scale bar, 20 μm. (C) The percentage of cells displaying Golgi-localized cPLA 2 α was determined from B and expressed relative to dimethyl sulfoxide (DMSO)–treated control. *p < 0.01; three independent experiments analyzing 200 cells. (D) Confluent HUVECs were pretreated with CytD (400 nM, 30 min) prior to biotinylation, lysis, and isolation of labeled proteins with NeutrAvidin agarose. Bound proteins were analyzed by immunoblotting. (E) Plasma membrane levels of VE-cadherin, VEGFR2, and TfR were quantified and expressed relative to control DMSO-treated cells. **p < 0.01; three independent experiments. (F) Confluent HUVECs were treated with CytD (400 nM, 30 min) or DMSO prior to lysis, preclearing, and immunoprecipitation with anti-PAR3 antibodies. Bound proteins were analyzed for VE-cadherin, β-catenin, AnxA1, PAR3, and cPLA 2 α.

Article Snippet: HUVECs were transfected with 6 pmol/13-mm 2 dish of nontargeting annealed control siRNA (Scrambled D-001210-01; Dharmacon, Lafayette, CO), annexin A1 (sc-29198, Santa Cruz Biotechnology, or 146987, Ambion, Austin, TX), cPLA 2 α (Ambion; Herbert et al. , 2009 ), α-catenin (sc-29190, Santa Cruz Biotechnology; or L-010505, Dharmacon), β-catenin siRNA (s436; Ambion), γ-catenin (sc-29324, Santa Cruz Biotechnology; or L-011708, Dharmacon), δ-catenin (sc-43021, Santa Cruz Biotechnology; or L-012572, Dharmacon), PAR3 (s32128, s32127, s32126; Ambion) or VE-cadherin siRNA (sc-36814, Santa Cruz Biotechnology; or s2780, Ambion) for 5–6 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

Techniques: Lysis, Isolation, Labeling, Western Blot, Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A 2 α in endothelial cells

doi: 10.1091/mbc.E11-08-0694

Figure Lengend Snippet: Inhibition of cPLA 2 α reduces HUVEC angiogenesis induced by PAR3 and α-catenin depletion. (A) HUVECs were grown overnight on Matrigel-coated coverslips prior to fixation and processing for cPLA 2 α (green), VE-cadherin (red), and TNG46 (purple) expression. Scale bar, 20 μm. (B) HUVECs were grown for 9 d upon a confluent monolayer of primary human foreskin fibroblasts prior to fixation and processing for confocal immunofluorescence microscopy analysis with the indicated antibodies. Scale bars, 20 μm. (C) HUVECs treated with indicated siRNA were grown for 9 d as in B, except that VEGF-A (25 ng/ml) and pyrrolidine (1.5 μM) were added after 4 d in culture. Cells were fixed and stained with anti-CD31 antibody and visualized by DAB staining. (D) Total tubule length per field and (E) average branch points per field were calculated and compiled from across three independent experiments performed in triplicate. *p < 0.05. (F) HUVECs treated with indicated siRNA were treated with A23187 in the presence or absence of pyrrolidine (1.5 μM). Released PGE2 was measured by enzyme-linked immunosorbent assay and expressed as a value of control unstimulated levels.

Article Snippet: HUVECs were transfected with 6 pmol/13-mm 2 dish of nontargeting annealed control siRNA (Scrambled D-001210-01; Dharmacon, Lafayette, CO), annexin A1 (sc-29198, Santa Cruz Biotechnology, or 146987, Ambion, Austin, TX), cPLA 2 α (Ambion; Herbert et al. , 2009 ), α-catenin (sc-29190, Santa Cruz Biotechnology; or L-010505, Dharmacon), β-catenin siRNA (s436; Ambion), γ-catenin (sc-29324, Santa Cruz Biotechnology; or L-011708, Dharmacon), δ-catenin (sc-43021, Santa Cruz Biotechnology; or L-012572, Dharmacon), PAR3 (s32128, s32127, s32126; Ambion) or VE-cadherin siRNA (sc-36814, Santa Cruz Biotechnology; or s2780, Ambion) for 5–6 h using Lipofectamine RNAiMAX transfection reagent (Invitrogen).

Techniques: Inhibition, Expressing, Immunofluorescence, Microscopy, Staining, Enzyme-linked Immunosorbent Assay