Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Patient sample characteristics: ibrutinib sensitivity, IgVH mutation status, diabetic status, Clinical Status, RAI stage, del13q14 status, and del17p status.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Compounds used to modulate CLL lymphocyte metabolism.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques: Concentration Assay

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: CLL lymphocytes display metabolic differences associated with ibrutinib sensitivity. (A–C) Survival fraction (relative to non-treated control [NT]) of CLL lymphocytes treated with metabolic inhibitors for 48 h, (A) ( n = 26), (B) ( n = 15–26), (C) ( n = 11), (mean ± SEM). Basal metabolite uptake differences between ibrutinib sensitive and resistant CLL lymphocytes, (D) Glucose ( n = 20), (E) Ammonia ( n = 22), (F) Glutamine ( n = 16), (G) Glutamate ( n = 23). Negative values indicate metabolite excretion to the media. (H) Basal raw (Resazurin) signal of mitochondrial reductive capacity of ibrutinib sensitive and resistant CLL lymphocytes ( n = 25). (I) Basal raw ROS (CellRox) signal of ibrutinib sensitive and resistant CLL lymphocytes ( n = 25). Basal protein expression of ibrutinib sensitive and resistant CLL lymphocytes (relative to actin signal) (J) GLUT1 ( n = 22) and (K) GLUT4 ( n = 10), * p < 0.05.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques: Control, Expressing

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Metabolic and Redox effects of ibrutinib on CLL lymphocytes. (A) Quantification of total glutathione after 24 h treatment with ibrutinib ( n = 15), (mean ± SEM). (B) Reduced/Oxidized glutathione ratio after 24 h treatment with ibrutinib ( n = 14). (C) NADPH/NADP ratio after 24 h of ibrutinib treatment ( n = 8) (mean ± SEM). (D) NADH/NAD ratio after 24 h of ibrutinib treatment ( n = 11) (mean ± SEM). Metabolite uptake after 24 h of ibrutinib treatment. (E) Glutamine ( n = 21) and (F) Glutamate ( n = 20) (mean ± SEM), * p < 0.05.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques:

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Inhibition of FAO decreases ibrutinib-induced cytotoxicity in CLL primary lymphocytes. (A) Survival fraction (relative to non-treated control) after 48 h treatment with ibrutinib alone or in combination with Fatty acids (FA), ( n = 10). (B) Survival fraction (relative to non-treated control) after 48 h treatment with etomoxir alone or in combination with ibrutinib, ( n = 12). (C) Total glutathione levels after 24 h treatment with etomoxir alone or in combination with ibrutinib ( n = 11). Protein expression levels (relative to actin) after 24 h treatment with etomoxir alone or in combination with ibrutinib (D) GS ( n = 11), (E) GAC ( n = 10), (I) GLUT1 ( n =10). (F) NADPH/NADP ratio ( n = 8), and (G) NADH/NAD ratio ( n = 11) after 24 h treatment with etomoxir alone or in combination with ibrutinib. (H) Relative ROS levels after 48 h treatment with etomoxir alone or in combination with ibrutinib ( n = 12), (mean ± SEM). (J) Glucose uptake after 24 h treatment with ibrutinib and etomoxir ( n = 8). * p < 0.05, ** p < 0.001.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques: Inhibition, Control, Expressing

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Effect of FAO inhibition in ibrutinib treated CLL lymphocytes positive for del17p. (A) Survival fraction (relative to non-treated control [NT]) after 48 h treatment with the indicated metabolic inhibitors ( n = 20–30) (mean ± SEM). (B) Basal NADH/NAD ratio ( n = 11), and (C) Basal NADPH/NADP ratio ( n = 14) of CLL lymphocytes negative and positive to del17p. (D) Reduced/Oxidized glutathione ratio ( n = 18), and (E) Basal total glutathione levels ( n = 19) of CLL lymphocytes negative and positive to del17p. (F) Basal raw ROS (CellRox) signal on CLL lymphocytes negative and positive to del17p ( n = 29). (G) Survival fraction (relative to non-treated control) after 48 h treatment with combinations of ibrutinib and etomoxir ( n = 4). wt -del17p negative CLL lymphocytes.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques: Inhibition, Control

Journal: Frontiers in Oncology

Article Title: Ibrutinib Resistance Is Reduced by an Inhibitor of Fatty Acid Oxidation in Primary CLL Lymphocytes

doi: 10.3389/fonc.2018.00411

Figure Lengend Snippet: Model for metabolic rewiring associated to ibrutinib resistance in CLL lymphocytes. Upon ibrutinib treatment, ibrutinib resistant cells initiate a compensation mechanism increasing fatty acid oxidation metabolism as well as α-KG production from glutamate, to maintain mitochondrial homeostasis. In line with this, we propose that α-KG follows preferentially an oxidation process in the TCA. Transporters are colored in purple, enzymes in green, and inhibitors in red. Enzymes overexpressed in CLL lymphocytes are in bold green characters. Blue arrows indicate reactions increased in basal ibrutinib resistant cells compared to the sensitive subset. Purple arrows represent the processes that are increased in resistant cells upon ibrutinib treatment. Dashed red arrows represent the processes inhibited upon ibrutinib treatment. Brown arrows indicate the proposed alternative metabolic rewiring to increase fatty acid and Acetyl-CoA availability.

Article Snippet: Cells were treated for 24 or 48 h with the following compounds: Ibrutinib (Selleckchem, S2680), Oligomycin A (Sigma, 75351), 2-Deoxy-D-glucose (Sigma, D6134), Ritonavir (Selleckchem, S1185), Compound 968 (Calbiochem, 352010), DHEA (Sigma, D-063), AMPA (Sigma, 324817), Etomoxir (Sigma, E1905), N-Acetyl-L-cysteine (NAC) (Sigma, A7250), Fatty acid supplement (Sigma, F7050).

Techniques:

Journal: Stem Cells International

Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

doi: 10.1155/2018/5034679

Figure Lengend Snippet: S1P receptor subtype expression and SphK/S1PR axis role in cell gelatinolytic activity. (a and c) Expression of S1P receptors by reverse transcription (RT) and real-time PCR analysis. mRNA were determined by RT of total RNA (1 μ g) obtained from BM-MSCs at low- (L-) and high- (H-) density culture and 2 μ l of cDNA (for S1PR1, S1PR2, and S1PR3 detection) or 4 μ l of cDNA (for S1PR4 and S1PR5 detection) were amplified as described in . Representative agarose gels of amplified DNA are shown. GAPDH amplification was used for data normalization. (b and d) Quantification of mRNA expression by real-time PCR analysis. Data are reported as mean ± S.E.M. of the ratio between the fold of variation of S1P receptor expression obtained from high- and low-density BM-MSCs culture. (e and f) BM-MSCs seeded onto fluorescein-labeled gelatin substrate- (DQ gelatin-) coated plastic culture plates (e) or glass coverslips (f) were cultured for 48 h in absence (vehicle) or in presence of the following compounds: 5 μ M sphingosine kinase inhibitor (iSK), 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871. (e) Spectrophotometrical quantification of the DQ gelatin fluorescence intensity revealed after proteolytic digestion of the gelatin by MMP gelatinases. (f) Representative superimposed DIC (grey) and fluorescent confocal microscopy images (green; gelatin fluorescence intensity) of fixed cells. Scale bar 30 μ m. Histogram shows the densitometric analysis of the intensity of the gelatin fluorescence signals performed on digitized images. Data reported as mean ± S.E.M. are representative of at least three independent experiments with similar results. Significance of differences in (b) and (d) (Student's t -test), ∗ p < 0.05 and ∗∗ p < 0.01; in (e) and (f) (one-way ANOVA and Newman-Keuls multiple comparison test), ∗ p < 0.05 versus vehicle, ° p < 0.05 versus SEW2871.

Article Snippet: The cells were plated at low- (3–5,000 cells cm −2 ) and high- (15–20,000 cells cm −2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

Techniques: Expressing, Activity Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Amplification, Labeling, Cell Culture, Fluorescence, Confocal Microscopy, Comparison

Journal: Stem Cells International

Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

doi: 10.1155/2018/5034679

Figure Lengend Snippet: MMP-2 expression and activity. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P) or 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative immunofluorescence confocal images of fixed cells on glass coverslips immunostained with antibodies against MMP-2 (green). Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. (b) Densitometric analysis of the intensity of the MMP-2 fluorescence signal performed on digitized images. (c) Zymography. A representative gelatin zymography of MMP-2 from conditioned media obtained from BM-MSCs incubated in absence (vehicle) or in presence of W146 or SEW2871 for 48 h. Densitometry scanning from at least three separate experiments was performed and data, expressed as relative OD values (a.u.) to those of control group (vehicle) set to 100, are reported in the histogram. Data are mean ± S.E.M. Significance of difference in (b) (one-way ANOVA and Newman-Keuls multiple comparison tests), ∗ p < 0.05 versus vehicle, ° p < 0.05 versus SEW2871; in (c) (Student's t -test), ∗ p < 0.05 versus vehicle.

Article Snippet: The cells were plated at low- (3–5,000 cells cm −2 ) and high- (15–20,000 cells cm −2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

Techniques: Expressing, Activity Assay, Cell Culture, Immunofluorescence, Fluorescence, Zymography, Incubation, Control, Comparison

Journal: Stem Cells International

Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

doi: 10.1155/2018/5034679

Figure Lengend Snippet: Cytoskeleton organization and cortactin expression. BM-MSCs were cultured for 48 h in the absence (vehicle) or in presence of the following compounds: 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, and 2 μ M S1PR1 receptor agonist, SEW2871 and/or MMP-2/9 inhibitor, SB-3CT (5 μ M or 10 μ M). (a–h) Representative immunofluorescence confocal images of cells cultured on glass coverslips in the indicated experimental conditions, fixed and stained with Alexa 568-phalloidin to visualize actin filaments (red) and immunostained with antibodies against cortactin (green). Scale bar 50 μ m. Arrows indicate filopodia and arrowheads indicate lamellipodia (L). (A–D) Magnifications of the indicated squared regions of interest showing the red and green fluorescence signals separately and together. Yellow-orange colour indicates colocalization between the two fluorescence signals. Scale bar 12 μ m. The images are representative of at least three independent experiments with similar results. (i) Densitometric analysis of the intensity of the cortactin fluorescence signal performed on digitized images. Data are mean ± S.E.M. Significance of differences (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p < 0.05 versus vehicle, ° p < 0.05 versus SEW2871.

Article Snippet: The cells were plated at low- (3–5,000 cells cm −2 ) and high- (15–20,000 cells cm −2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

Techniques: Expressing, Cell Culture, Immunofluorescence, Staining, Fluorescence, Comparison

Journal: Stem Cells International

Article Title: Sphingosine 1-Phosphate Receptor 1 Is Required for MMP-2 Function in Bone Marrow Mesenchymal Stromal Cells: Implications for Cytoskeleton Assembly and Proliferation

doi: 10.1155/2018/5034679

Figure Lengend Snippet: Cell proliferation and toxicity. BM-MSCs were incubated in growth medium for 24 h in absence (vehicle) or in presence of 1 μ M exogenous sphingosine-1-phosphate (exoS1P), 2 μ M S1PR1 receptor antagonist, W146, or 2 μ M S1PR1 receptor agonist, SEW2871. (a) Representative confocal immunofluorescence images of Ki67 expression. BM-MSCs were immunostained with the specific antibody Ki67 (green), a nuclear proliferation marker, and counterstained with propidium iodide (PI; red). Yellow colour indicates colocalization of red and green fluorescence signals. Scale bar 50 μ m. The images are representative of at least three independent experiments with similar results. Histogram represents quantitative analysis of Ki67 positive BM-MSC cell nuclei expressed as percentage of the total nuclei number. Data are mean ± S.E.M. (b) Cell proliferation analysis by cell counting. Synchronized BM-MSCs were collected and counted as reported in . Data are mean ± S.E.M. of four independent experiments performed in quadruplicate. (c) Western blotting analysis of apoptotic (Bax) and autophagic (Beclin) markers. Cell lysates (10–25 μ g) obtained from BM-MSCs were loaded onto SDS-PAGE and proteins immunodetected by specific antibodies. β -Actin was used as loading control. Blot shown is representative of at least three independent experiments with similar results. Data resulting from densitometric analysis of at least three independent experiments are shown in the graph (mean ± S.E.M.). Significance of differences in (a) and (b) (one-way ANOVA and Newman-Keuls multiple comparison test): ∗ p < 0.05 versus vehicle.

Article Snippet: The cells were plated at low- (3–5,000 cells cm −2 ) and high- (15–20,000 cells cm −2 ) density confluence and treated for 48 h with specific vehicle or with the following compounds: sphingosine kinase inhibitor (iSK, 5 μ M, Tocris Bioscience, Bristol, UK) [ , , ]; sphingosine 1-phosphate (S1P, 1 μ M, Calbiochem, San Diego, CA, USA, stock solution 2 mM in DMSO) S1PR1 receptor antagonist, W146 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], S1PR1 receptor agonist, SEW2871 (2 μ M, Tocris Bioscience, stock solution 2 mM in DMSO) [ ], MMP-2, and MMP-9 inhibitor, SB-3CT (5 and 10 μ M, Sigma, Milan, Italy, stock solution 10 mM in DMSO) [ ].

Techniques: Incubation, Immunofluorescence, Expressing, Marker, Fluorescence, Cell Counting, Western Blot, SDS Page, Control, Comparison

Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 4 PGK1 plays an essential role in BoHV-1 productive infection. A MDBK cells in 6-well plates were transfected with either scrambled siRNA (200 pmol) or three individual siRNA targeting PGK1 (200 pmol), referred to as siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 48 h post-transfection, PGK1 protein levels were detected via western blot. B, C MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA, siRNAPGK1-1, and siRNAPGK1-2, respectively. After transfection for 36 h, the cells were infected with BoHV-1 (MOI = 0.1) for one h. After three washes with PBS, the fresh medium was replaced. At 12 and 24 hpi virus yield in the cell cultures was measured, with results expressed as TCID50/mL (B), and levels of viral DNA were examined from intracellular content using qPCR with gC-specific primers (C). D The cytotoxicity of NG52 (5μM) in MDBK cells for 24 h was analysed by Trypan-blue exclusion test. E and F MDBK cells in 24-well plates pretreated with either DMSO control or NG52 at indicated concentrations were infected with BoHV-1 (MOI = 0.1) for one hour along with treatment-indicated chemicals. After three washing with PBS, fresh media containing DMSO control or NG52 were added for further incubation. At 12 and 24 hpi, the virus titers were measured, with the results expressed as TCID50/mL (E), and the intracellular content of virus genomic DNA was determined using relative qPCR with gC-specific primers (F). G and H MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA or siRNAPGK1-1. After transfection for 36 h, they were infected with BoHV-1 (MOI = 0.1) for 24 h. Total RNA was purified, and mRNA levels of bICP27 (G) and VP16 (H) were subsequently detected via qRT-PCR, respectively. The results shown are the means of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (* p < 0.05; ns = insignificant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Infection, Transfection, Western Blot, Virus, Control, Incubation, Purification, Quantitative RT-PCR

Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 6 PGK1 positively regulates β-catenin expression. A MDBK cells in 6-well plates were treated with either DMSO control or NG52 at indicated concentrations for 24 h. The cell lysates were prepared and subjected to western blot to detect β-catenin protein levels. C MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA, siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 48 h after transfection, β-catenin protein levels were detected by western blot. E PGK1 plasmid along with empty vector at the indicated dose were transfected into Neuro-2A cells in 6-well plates using lipofectamine 3000; after transfection for 48 h, the cells were either collected for the detection of β-catenin protein levels via western blot. G MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA or siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 36 h after transfection, the cells were infected with BoHV-1 at an MOI of 0.1 for 24 h. Cell lysates were prepared with RIPA buffer and then protein levels of β-catenin were analysed using western blotting. GAPDH was probed as a loading control. B, D, F, and H Band intensity was analysed using the Image J software. The control was arbitrarily set as either 1 or 100%. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (*p < 0.05; ns = not significant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Infection, Software

Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 7 PGK1 stimulates β-catenin-dependent transcription. A MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1(MOI = 0.1) for 24 h. Cell lysates were subjected to IP using antibodies against either PGK1 β-catenin or isotype IgG. Then both β-catenin and PGK1 were detected by western blot. The data shown are representative of three independent experiments. B Neuro-2A cells were co-transfected with 0.1 μg of the Super 8 × TOPFlash luciferase reporter construct, 0.01 μg of the Renilla reporter construct, and 0.25 μg of β-cateninS33Y mutant (S33Y) plasmid, together with increasing concentrations of a plasmid expressing PGK1 (0.5 or 1 μg) to examine the effect that PGK1 has on TCF promoter activity. At 48 h after transfection, dual luciferase assays were performed. C MDBK cells in 12-well plates (60% confluent) were transfected with 0.4 μg of the Super 8 × TOPFlash luciferase reporter construct and 0.05 μg of the Renilla reporter construct that was used as an internal control to allow normalisation of promoter activity. After transfection for 36 h, the cells were infected with BoHV-1 (MOI = 1) for 24 h along with treatment of either DMSO control or NG52 at the designated concentrations. Dual luciferase assays were performed 24 h after infection. The results shown are the average of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (*p < 0.05; ns, not significant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Infection, Western Blot, Transfection, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay, Control