Journal: Virology

Article Title: Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

doi: 10.1016/j.virol.2021.10.009

Figure Lengend Snippet: Fig. 3. Inhibition of ERK1/2 activation impairs SADS-CoV infection. (A) U0126 and PD98059 treatments do not affect cell viability. Vero E6 and IPI-2I cells were treated with U0126 or PD98059 at different concentrations or with the vehicle control DMSO for 36 h. Cell cytotoxicity was analyzed with the CCK-8 kit, as described in the Materials and Methods. (B) SADS-CoV propagation in the presence of U0126 or PD98059. Vero E6 cells were pretreated with either inhibitor at the indicated concentrations for 1 h and then infected with SADS-CoV. SADS-CoV-infected cells were further incubated for 36 h in the presence of DMSO, U0126, or PD98059. At 36 hpi, the virus-infected cells were subjected to an immunofluorescence assay with an anti-SADS-CoV-N antibody, followed by DAPI counterstaining and exami nation under an inverted fluorescence microscope. The percentage of SADS-CoV-infected cells per view is expressed as the mean ± SD of three independent ex periments. (C and D) Viral N protein expression in the presence of U0126 or PD98059. Vero E6 and IPI-2I cells were treated with DMSO, U0126, or PD98059 at the indicated concentration for 1 h before infection with SADS-CoV. The SADS-CoV-infected cells were then incubated for a further 36 h in the presence of DMSO, U0126, or PD98059. At 36 hpi, the cell lysates were examined with western blotting, probed with an antibody directed against SADS-CoV N protein. The blot was also reacted with a mouse monoclonal antibody directed against GAPDH to verify equal protein loading. Densitometric data for SADS-CoV N/GAPDH are expressed as the means ± SD of three independent experiments. (E and F) U0126 or PD98059 treatment suppressed SADS-CoV replication. Treatment and infection conditions were as described for panels C and D. The viral titers in the supernatants collected at 36 hpi were determined with the Spearman–K¨arber method. Error bars represent the standard errors of the means of three independent experiments.

Article Snippet: U0126 and PD98059 were obtained from Cell Signaling Technology (Danvers, USA).

Techniques: Inhibition, Activation Assay, Infection, Control, CCK-8 Assay, Incubation, Virus, Immunofluorescence, Fluorescence, Microscopy, Expressing, Concentration Assay, Western Blot

Journal: Virology

Article Title: Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway.

doi: 10.1016/j.virol.2021.10.009

Figure Lengend Snippet: Fig. 5. U0126 blocks SADS-CoV-induced apoptosis. (A) Vero E6 cells were mock infected or infected with SADS-CoV (MOI = 0.1) in the presence or absence of U0126 (10 μM) for 36 h. They were washed twice with cold PBS and resuspended in 1 × binding buffer. The cells were stained with FITC–annexin V and PI for 15 min and analyzed with flow cytometry (BD FACSCalibur, USA) within 1 h. Q1: necrotic or other cell population, which was FITC–annexin V negative and PI positive; Q2: end-stage apoptotic or dead cell population, which was FITC–annexin V and PI positive; Q3: early apoptotic cell population, which was FITC–annexin V positive and PI negative; Q4: viable cell population that was not undergoing apoptosis, which was both FITC–annexin V and PI negative. The graph on the right represents the percentage of each cell population nonsignificant percentages of annexin-V-negative and PI-positive cells were excluded. (B and C) Vero E6 and IPI-2I cells were mock infected or infected with SADS-CoV at an MOI of 0.1 in the presence or absence of U0126 (10 μM). After incubation for 36 h, a western blotting analysis with antibody specific for p-ERK1/2, ERK1/2, PARP, or SADS-CoV N protein. GAPDH was used as the internal loading control. (D) The virus-containing supernatants were collected at 36 hpi and the viral titers were calculated with the Spearman–K¨arber method. Error bars represent the standard errors of the means of three independent experiments.

Article Snippet: U0126 and PD98059 were obtained from Cell Signaling Technology (Danvers, USA).

Techniques: Infection, Binding Assay, Staining, Flow Cytometry, Incubation, Western Blot, Control, Virus

Journal: Microorganisms

Article Title: Three Lipid Emulsions Reduce Staphylococcus aureus -Stimulated Phagocytosis in Mouse RAW264.7 Cells

doi: 10.3390/microorganisms9122479

Figure Lengend Snippet: The activation of PI3K and JNK is involved in the lipid emulsion-caused reduction in phagocytosis. ( a ) The increase of p-JNK in S. aureus -infected RAW264.7 cells during the indicated time points was detected by western bolt analysis. ( b ) The phosphorylation of AKT, JNK, and ERK in S. aureus -infected RAW264.7 cells with or without lipid emulsion treatment was determined by western blot analysis. ( c ) The inhibitors’ effects on the phagocytosis of RAW264.7 cells were quantified by pHrodo™ Green S. aureus Bioparticles ® conjugate in the absence (positive control) or presence of indicated reagents. The reagents included the inhibitors of PI3K (LY294002), JNK (SP600125), ERK (PD98059), and phagocytosis (cytochalasin D). ( d ) Image of phagocytosis in live cells. RAW264.7 cells were treated with and without inhibitors prior to and during phagocytosis. The nuclei of cells were stained with Hoechst 33342 (blue). The location of the lysozymes was indicated by LysoTracker (red). The images were obtained by confocal microscopy with a 10× objective. ( e ) The inhibitors induced the changes in F-actin polymerization. Alexa-Fluor 568-labeled phalloidin was used to visualize the F-actin of S. aureus -infected RAW264.7 cells in the absence or presence of inhibitors, which was observed by confocal microscopy (63×). White arrow = filopodia. ( f ) Treatment with PI3K inhibitors significantly increased S. aureus survival. S. aureus -infected RAW264.7 cells were treated with or without the indicated reagent. At the start of infection (0 h), the cell-associated colony-forming units (CFUs) were similar between different treatments. Bacterial survival CFUs were grown and counted at the end of infection (3 h). The data are shown as the means ± SD of three individual experiments. Significance was analyzed by one-way ANOVA followed by the Bonferroni test ( p < 0.05). * Significant difference compared with the S. aureus -infected control.

Article Snippet: PD98059, a specific inhibitor of mitogen-activated protein kinase, was purchased from TargetMol (Boston, MA, USA).

Techniques: Activation Assay, Infection, Western Blot, Positive Control, Staining, Confocal Microscopy, Labeling

Journal: Frontiers in Immunology

Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

doi: 10.3389/fimmu.2018.01517

Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

Article Snippet: To assess PAR-induced cell signaling, starved cells were treated with 0–20 µM of either PAR-1, PAR-2, or PAR-4 antagonists (Peptides International, Louisville, KY, USA) for 30 min before stimulation with FVIIa + FX or FVIIa + FX + FII at indicated doses; or cells were stimulated with 0–100 µM of PAR-1, PAR-2, or PAR-4 agonist (Peptides International); or cells were treated first with or without 50 µM mitogen-activated protein kinase inhibitor PD98059, 10 µM p38-MAPK inhibitor SB203580, 20 µM NF-kB inhibitor SN50, and 1 µM of the S6K1 inhibitor (All from Merck Millipore, Hertfordshire, UK) for 30 min and then stimulated with 10 µM of PAR-1, PAR-2, or PAR-4 agonist.

Techniques: Coagulation, Isolation, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY

Journal:

Article Title: The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways

doi: 10.1038/nature03966

Figure Lengend Snippet: A) Diagram depicting the modular composition and action of AKAR2. B) Diagram of AKAR2-PKA. Bottom) Pseudo-coloured images of FRET changes in HeLa cells stimulated with cAMP over a 15 min time course. C) Diagram of AKAR2-PKA-PDE. Bottom) Pseudo-coloured images of FRET changes in HeLa cells stimulated with cAMP over a 15 min time course. D) Amalgamated FRET measurements for AKAR-PKA (Red, n=10), AKAR-PKA-PDE (Green, n=13), AKAR-PKA + H89 (Blue, n=10) and AKAR-PKA-PDE + H89 (Black, n=7) for 15 minutes after cAMP stimulation with forskolin (arrow). E) Amalgamated FRET traces (n=10) using the AKAR-PKA-PDE reporter after application of the PDE3 inhibitor milrinone (0–12 min) and the PDE4 inhibitor rolipram (24–36 min). Stimulation with cAMP was at times 0 and 24 min (arrows). F) FRET measurements from HeLa cells expressing the AKAR-PKA-PDE reporter in the presence (black, n=11) or absence (green, n=7) of a dominant active MEK5 for 15 minutes after cAMP stimulation with forskolin (arrow). G) Phosphodiesterase activity (n=4, error bars show S.E.M.) in mAKAP immune complexes isolated from RNVs. Treatment conditions are indicated above each column. H) ERK activity in the mAKAP complex from heart extracts (n=3, error bars show S.E.M.). Treatments with kinase inhibitors are indicated. I) Co-precipitated ERK was detected by immunoblot using (top) pan-ERK, (middle) ERK5 specific and (bottom) MEK5 specific antibodies. P values <0.01 (**) are indicated relative to control (black) and sample (red).

Article Snippet: Antibodies Primary antibodies were: monoclonal pan-ERK (Transduction Labs), monoclonal MEK5 (Transduction Labs); monoclonal pan-PDE4D (ICOS); rabbit ERK5 (StressGen); monoclonal 9E10-Myc (Upstate); rabbit mAKAP (VO54); rabbit Epac1 (Santa Cruz).

Techniques: Expressing, Activity Assay, Isolation, Western Blot, Control

Journal:

Article Title: The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways

doi: 10.1038/nature03966

Figure Lengend Snippet: A) Serum stimulated mAKAP associated ERK5 activity (n=3, error bars show S.E.M.) in parallel cultures pretreated with forskolin to elevate cAMP (columns 2&3) or in the presence of the PKA inhibitors H89, KT5720, or Rp-cAMPs (columns 4–6). The amount of ERK5 was detected in each sample. B) Immunoblot detection of Epac1 in mAKAP immune complexes from rat heart extracts. C–E) Fluorescent staining of hypertrophic RNV with antibody for Epac1 (C) and Alexa 568 phalliodin for the actin cytoskeleton (D). Composite image (E) shows the distribution of Epac1 (green) and actin (red, scale bar =20μm). F) The mAKAP complex was immunoprecipitated from cultured RNV following serum stimulation to activate ERK. Parallel cultures were pretreated with either the Epac-selective activator 007 or KT5720 prior to serum stimulation (n=3, error bars show S.E.M.). G & H) Serum stimulated mAKAP associated ERK activity in cells expressing of constitutively active RapGAP (J, n=4, error bars show S.E.M.) or control β-galactosidase (K, n=3, error bars show S.E.M.). Stimulation of intracellular cAMP or treatment with the kinase inhibitor KT5720 is indicated. P values <0.01 (**) are indicated relative to control (black) and sample (red).

Article Snippet: Antibodies Primary antibodies were: monoclonal pan-ERK (Transduction Labs), monoclonal MEK5 (Transduction Labs); monoclonal pan-PDE4D (ICOS); rabbit ERK5 (StressGen); monoclonal 9E10-Myc (Upstate); rabbit mAKAP (VO54); rabbit Epac1 (Santa Cruz).

Techniques: Activity Assay, Western Blot, Staining, Immunoprecipitation, Cell Culture, Expressing, Control

Journal:

Article Title: The protein kinase A anchoring protein mAKAP co-ordinates two integrated cAMP effector pathways

doi: 10.1038/nature03966

Figure Lengend Snippet: A–C) Leukemia inhibitory factor (LIF) induced changes in RNV size. The outline of RNV from control (A) and LIF treated (B) samples are presented, scale bar =20μm. C) Quantitation of cell size (μm2) in control (1) and LIF treated RNV (2–5). Pharmacological manipulation of ERK5 (PD98095), Epac1 (007) or PDE4 (rolipram) activity is indicated (error bars show S.E.M.). The number of experiments is indicated above each column. D) Expression of mAKAP was suppressed by RNA interference. Quantitation of LIF induced hypertrophy (cell size +LIF/−LIF) in control (1), mAKAP silenced (2) and RNV rescued with an mAKAP form resistant to the shRNA (3, error bars show S.E.M.). E) Displacement of mAKAP from the nuclear membrane was achieved by overexpression of the mAKAP targeting domain fragment using the TET OFF inducible promoter. Quantitation of cell size (μm2) from control (1), from LIF-stimulated RNV controls (2, TET promoter OFF), and from LIF-stimulated cells expressing the mAKAP 585–1286 fragment (3, TET promoter ON, error bars show S.E.M.). F–I) Schematics depicting the major findings of this study. F) ERK5 phosphorylation of PDE4D3 shuts down cAMP metabolism. G) PKA phosphorylation of PDE4D3 enhances cAMP metabolism. H) Activation of Epac mobilizes Rap1 to suppress ERK5 activation, and I) low cAMP represses the Epac mediated block of ERK5 allowing cardiac hypertrophy. P values <0.01 (**) and p values <0.05 (*) are indicated relative to control (black) and sample (red).

Article Snippet: Antibodies Primary antibodies were: monoclonal pan-ERK (Transduction Labs), monoclonal MEK5 (Transduction Labs); monoclonal pan-PDE4D (ICOS); rabbit ERK5 (StressGen); monoclonal 9E10-Myc (Upstate); rabbit mAKAP (VO54); rabbit Epac1 (Santa Cruz).

Techniques: Control, Quantitation Assay, Activity Assay, Expressing, shRNA, Membrane, Over Expression, Phospho-proteomics, Activation Assay, Blocking Assay