Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of immunotoxicology

Article Title: The early growth response factor-1 contributes to interleukin-13 production by mast cells in response to stem cell factor stimulation.

doi: 10.1080/15476910802129612

Figure Lengend Snippet: FIG. 4. Egr-1 deficiency has no effects on c-Kit and IgE receptor expression or mast-cell viability. (A) Bone marrow cells from wild-type mice or Egr-1– deficient mice were cultured in conditioned media in vitro for 4 wk and exam- ined by flow cytometry for c-Kit and IgE receptor expression. For c-Kit anal- ysis, BMMCs were stained with FITC conjugated rat anti-mouse c-Kit mAb or FITC conjugated rat IgG2a isotypic control. For analysis of IgE receptor expression, BMMC were sensitized with IgE overnight and then stained with FITC-conjugated anti-IgE antibody (mouse IgG1). No difference in c-Kit or IgE receptor expression was observed between Egr-1+/+ and Egr-1−/−BMMCs. (B) BMMC from Egr-1-deficient mice or wild-type mice cultured in complete media containing WEHI-3B supernatants (a source of IL-3) were healthy and showed ≥96% viability. To induce mast cell death, WEHI-3B supernatant was removed from culture media for various days. Mast cell viability was examined by trypan blue exclusion assay. Data were expressed as mean ± SD (n = 3 independent experiments).

Article Snippet: Fluorescein isothiocyanate (FITC) anti-mouse CD117 monoclonal antibody (mAb) (CL8936F), FITC rat IgG2a (CLCR2A01) were purchased from Cedarlane Laboratories Limited (Ontario, Canada).

Techniques: Expressing, Cell Culture, In Vitro, Cytometry, Staining, Control, Trypan Blue Exclusion Assay

Journal: PLoS ONE

Article Title: Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

doi: 10.1371/journal.pone.0166710

Figure Lengend Snippet: A) Tyrosine phosphorylation of the high-MW Gab1 was analyzed by immunoprecipitation of the heart lysates. Mouse heart lysates were prepared at 5 min after injection with the cytokines and growth factors listed at top. Heart lysates were subjected to immunoprecipitation with anti-high-MW Gab1 serum, followed by western blot analysis using the antibodies indicated at the left. Arrowheads denote the high-MW Gab1. B) Activation level of ErbB4 was assessed by phospho-specific antibody. C) Activation levels of ERK1/2 and AKT were assessed by phospho-specific antibodies. Heart lysates were subjected to western blot analysis with the indicated antibodies. Representative blots of 3 experiments are shown. D) Phosphorylation of ERK1/2 was quantified against total ERK1/2 (n = 3). E) Phosphorylation of AKT on Ser-473 was quantified against total AKT (n = 3). Values are shown as means±SEM for 3 separate experiments. One-way ANOVA followed by Tukey’s test was used to analyze differences. * P <0.05, ** P <0.01, and *** P <0.001 for the indicated groups.

Article Snippet: Other antibodies were purchased as follows: anti-phospho-Gab1 (Tyr627) (1:1000, #3231), anti-Gab1 (1:1000, #3232), anti-phospho-AKT (Ser473) (1:1000, #9271), anti-AKT (1:1000, #9272), anti-phospho-p44/p42 (pERK1/2) (1:1000, #9101), anti-p44/p42 (ERK1/2) (1:1000, #9102), anti-pErbB4 (Tyr1284) (1:1000, #4757) antibodies for immunoblotting, and horseradish peroxidase-coupled horse anti-mouse (1:3000, #7076) and goat anti-rabbit IgG (1:3000, #7074) from Cell Signaling Technology; antibodies recognizing phospho-tyrosine (PY99) (1:1000, sc-7020), ErbB4 (1:400, sc-283), and SHP2 (1:400, sc-280) were from Santa Cruz Biotechnology Inc; anti-Gab1 (1:1000, 06–579) antibody for immunoblotting of immunoprecipitated proteins, and anti-p85 (1:1000, 06–195) antibody were from Millipore; antibodies against β-tubulin (mouse monoclonal) (1:3000, T5201), and anti-α-actinin (1:300, A7732) were from Sigma-Aldrich.

Techniques: Phospho-proteomics, Immunoprecipitation, Injection, Western Blot, Activation Assay

Journal:

Article Title: Bone-Marrow-Derived Cells Contribute to Glomerular Endothelial Repair in Experimental Glomerulonephritis

doi:

Figure Lengend Snippet: Immunofluorescent double-staining of glomerular sections from WRBM→BN rats at 28 days after induction of anti-Thy-1.1 nephritis. Confocal laser-scanning microscopy (×630, pinhole 2.5). A: Staining for BM donor (WR) MHC-I (U9F4, green). B: Staining for endothelial cells (RECA-1, red). C: U9F4/RECA-1 double-staining identifies bone-marrow-derived endothelial cells, randomly integrated in the glomerular endothelium (U9F4/ED-1 double-positive, yellow). D: Staining for BM donor (WR) MHC-I (U9F4, green). E: Staining for mesangial cells (OX-7, red). F: U9F4/OX-7 double-staining showing donor bone-marrow-derived mesangial cells (U9F4/OX-7 double-positive, yellow) integrated in the glomerulus. G: Staining for BM donor (WR) MHC-I (U9F4, red). H: Staining for monocytes/macrophages (ED-1, green). I: U9F4/ED-1 double-staining shows a BM donor-derived (U9F4/ED-1 double positive, yellow) monocyte/macrophage. ▵▵, U9F4/OX-7, U9F4/RECA-1, U9F4/ED-1 double-positive cells (yellow), ie, donor-derived mesangial and endothelial cells and a donor-derived monocyte/macrophage. ∧∧, single-U9F4 staining.

Article Snippet: The following primary antibodies were used: fluorescein isothiocyanate (FITC)-labeled anti-BN-MHC-I (CL027F; Cedarlane, Hornby, Ontario, Canada), anti-WR-MHC-I (U9F4 19 ; kindly provided by J. Rozing, Groningen, the Netherlands), and secondary antibody goat anti-mouse-phycoerythrin (PE) (Southern Biotechnologies, Birmingham, AL).

Techniques: Double Staining, Confocal Laser Scanning Microscopy, Staining, Derivative Assay

Journal:

Article Title: Bone-Marrow-Derived Cells Contribute to Glomerular Endothelial Repair in Experimental Glomerulonephritis

doi:

Figure Lengend Snippet: Strain-Specific MHC-I Expression on Peripheral Blood Leukocytes of WR, BN, and WR BM → BN Rats

Article Snippet: The following primary antibodies were used: fluorescein isothiocyanate (FITC)-labeled anti-BN-MHC-I (CL027F; Cedarlane, Hornby, Ontario, Canada), anti-WR-MHC-I (U9F4 19 ; kindly provided by J. Rozing, Groningen, the Netherlands), and secondary antibody goat anti-mouse-phycoerythrin (PE) (Southern Biotechnologies, Birmingham, AL).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Alterations in Bone and Erythropoiesis in Hemolytic Anemia: Comparative Study in Bled, Phenylhydrazine-Treated and Plasmodium -Infected Mice

doi: 10.1371/journal.pone.0046101

Figure Lengend Snippet: (A–C) Blood was recovered from naive, bled, P.c. adami- infected and PHZ-treated female BALB/c mice prior to and every day following treatment for assessment of hemoglobin (Hb) and percentages of reticulocytes (CD71 + ). (D) Kinetics of parasitemia in P. c. adami BALB/c mice. (E) The lowest Hb concentrations (g/dL) and (F) highest reticulocytosis measured in mice are represented. Data are mean ± SEM from 4–7 mice per group, compiled and compared using a one–way ANOVA and Tukey's multiple test. * P<0.05, **P<0.01, ***P<0.001 are comparisons to naive; εε P <0.01, εεε P <0.001 are comparisons to bled; ψ P <0.05, ψψψ P <0.001 are comparisons between PHZ-treated and Plasmodium- infected mice.

Article Snippet: The cell suspensions were then stained with anti-CD71-FITC antibody (Cedarlane, Canada) and samples from Plasmodium -infected mice were additionally treated with propidium iodide (PI) (1 mg/mL stock) to exclude parasitized RBC from the analysis.

Techniques: Infection

Journal: PLoS ONE

Article Title: Alterations in Bone and Erythropoiesis in Hemolytic Anemia: Comparative Study in Bled, Phenylhydrazine-Treated and Plasmodium -Infected Mice

doi: 10.1371/journal.pone.0046101

Figure Lengend Snippet: Fresh bone marrow cell suspensions from the tibia of naive, bled, P.c. adami -infected and PHZ-treated female BALB/c mice (4–6 weeks old) were stained with anti-CD71-FITC and anti-Ter119-PE antibody for analysis of stage specific erythroblasts. Ter119 + cells were gated in R1 whereas a R2 region selected proerythroblasts (A). Cells within the R1 gate (Ter119 high) were further analyzed with respect to their CD71 and forward scatter (FSC) profiles (B) and the percentages of proerythroblasts (C), basophilic erythroblasts (R3) (D), basophilic/polychromatic erythroblasts (R4) (E) and late polychromatophilic erythroblasts (R5) (F) were estimated. Data are mean ± SEM from n = 4–5 mice per group and are compared to naive mice ( *P < 0.05, ** P <0.01, *** P <0.001), bled mice ( ε P <0.01, εεP <0.01, εεεP <0.001) or between Plasmodium- infected and PHZ-treated mice ( ΨΨΨ P <0.001) using a one– way ANOVA and Tukey's multiple test.

Article Snippet: The cell suspensions were then stained with anti-CD71-FITC antibody (Cedarlane, Canada) and samples from Plasmodium -infected mice were additionally treated with propidium iodide (PI) (1 mg/mL stock) to exclude parasitized RBC from the analysis.

Techniques: Infection, Staining

Journal: PLoS ONE

Article Title: T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice

doi: 10.1371/journal.pone.0075166

Figure Lengend Snippet: (A) Splenocytes from 4-mo-old mice were stained to assess the proportion of Tfh (CD4 + CD44 hi CD62L lo CXCR5 hi PD1 hi ) cells. Representative contour plots from B6 and c1(70-100) mice. Thick boxes denote the regions that were used to identify Tfh cells. Cells shown in the right panels were gated on the regions shown in the left panels. (B) Scatter plots showing the proportion of Tfh cells within the CD4 + T cell subset and absolute number of splenic Tfh cells. (C) Representative contour plots and histograms from flow cytometry analysis of IL-17-, IFN-γ-, and IL-4-expressing CD4 + T cells in B6 and c1(70-100) mice. Splenocytes were stimulated with PMA and ionomycin in the presence of GolgiStop for 4 h, and then fixed, stained with anti-CD3 and -CD4, permeabilized, and stained with anti-cytokine Ab. Thick lines outline the regions used to gate CD4 + CD3 + T cells. For histograms, the percentage of cells staining positively for each cytokine is indicated. (D) Scatterplots showing the percentages of cytokine-producing cells as a proportion of the CD4 + T cell population. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with **p<0.01, ***p<0.001. Bars with p values above denote significant differences between congenic strains.

Article Snippet: Biotin-conjugated peanut agglutinin (PNA) was purchased from Sigma-Aldrich and FITC-conjugated anti-CD62L (MEL-14) mAb was purchased from Cedarlane Laboratories (Burlington, ON, Canada).

Techniques: Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: T Cell and Dendritic Cell Abnormalities Synergize to Expand Pro-Inflammatory T Cell Subsets Leading to Fatal Autoimmunity in B6.NZBc1 Lupus-Prone Mice

doi: 10.1371/journal.pone.0075166

Figure Lengend Snippet: 8-wk-old mice were injected i.p. with OVA or PBS in CFA. The proportions of splenic Tfh cells (CD4 + CD44 hi CD62L lo CXCR5 hi PD1 hi ) and splenic GC B cells (B220 + Fas + PNA hi ) were determined by flow cytometry 2 wks later. (A) Scatterplots showing the proportion of Tfh and GC B cells as a proportion of the CD4 + T cell and B220 + B cell populations, respectively. (B) Scatterplots showing the amount of cytokine produced by OVA-primed splenocytes re-stimulated in-vitro with OVA for 72h. Assays were performed in triplicate and the levels of secreted cytokines measured by ELISA or cytokine bead array (see Methods). Each data point represents the mean of the triplicate with background cytokine production in the absence of antigen subtracted. Horizontal lines indicate the mean of each group examined. Significance levels were determined by one-way ANOVA with Dunns’ post-test. The p values for significant differences between B6 and congenic mouse strains are shown with *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Biotin-conjugated peanut agglutinin (PNA) was purchased from Sigma-Aldrich and FITC-conjugated anti-CD62L (MEL-14) mAb was purchased from Cedarlane Laboratories (Burlington, ON, Canada).

Techniques: Injection, Flow Cytometry, Produced, In Vitro, Enzyme-linked Immunosorbent Assay