Journal: BMC Medicine

Article Title: Neutrophil degranulation and severely impaired extracellular trap formation at the basis of susceptibility to infections of hemodialysis patients

doi: 10.1186/s12916-022-02564-1

Figure Lengend Snippet: Massive neutrophil degranulation and inadequate gene expression of HD neutrophils. A Western blot analysis of steady-state levels of the granules proteins MMP-9, MPO, and NE from highly pure neutrophil pellets taken from HC and HD donors . Total proteins were extracted from 3 HC or HD donors; GAPDH was used as a loading control. B Elevated levels of neutrophil degranulation surface markers CD18, CD11b, CD66b, and CD15 in HD compared to HC neutrophils. Overlay flow cytometry histogram plots of individual representative HC (blue) and HD (red) neutrophils derived from scatter plots showing levels of CD66b or CD15 (top panel). Mean fluorescence intensities of CD18, CD11b, CD66b, and CD15 in HD compared to HC donors (lower panel, n =4). C PCA analysis of RNA-Seq expression profile of 7 HD and 7 HC patients. D P -value and fold change (log2) filters for differential gene expression analysis of HC Vs. HD samples. HD upregulated genes are marked in red, and downregulated genes in blue. E Heatmap of selected gene set with neutrophil activation and NETosis-related functional annotation found in HD up-reg. Dataset. Expression levels are normalized between 0 and 1. F Normal differential expression distribution on five major NETosis functional genes showing a significantly higher expression in HD samples compared to HC donors. G Gene set enrichment analysis (GSEA) annotations and corresponding p -values found HD upregulated gene set to be enriched in neutrophil activation and degranulation-related genes

Article Snippet: Membranes were blocked with 5% BSA, incubated with specific antibodies against human PAD4 (ab96758; Abcam), Anti-Neutrophil Elastase (G-2; SC-55549; Santa Cruz Biotechnology, Inc.), Anti-MPO/Myeloperoxidase (266-6K1; SC-52707; Santa Cruz Biotechnology, Inc.), MMP-9 (2C3; SC-21733; Santa Cruz Biotechnology, Inc.), and the housekeeping protein GAPDH (Cell Signaling 2118S) as a loading control.

Techniques: Gene Expression, Western Blot, Control, Flow Cytometry, Derivative Assay, Fluorescence, RNA Sequencing, Expressing, Activation Assay, Functional Assay, Quantitative Proteomics

Journal: Environmental research

Article Title: Investigation of estrogen-like effects of parabens on human neutrophils.

doi: 10.1016/j.envres.2022.113893

Figure Lengend Snippet: Fig. 6. Effect of parabens and 17β-estradiol (E2) to the expression of estrogen receptor α (ERα), estrogen receptor β (ERβ) and G-protein coupled estrogen receptor 1 (GPER). The expression of ERα, ERβ and GPER was assessed in Western blot method in whole-cells lysates from female and male neutrophils. Cells were 1-h pre- incubated with ERα antagonist (MPP; 5 μM), ERβ antagonist (PHTPP; 5 μM) or GPER antagonist (G15; 5 μM), then, 2-h incubated with MeP, EtP, PrP, BuP, or E2 (10 nM). Relative band intensity of ERα, ERβ and GPER was quantified using ImageJ software and expressed in arbitrary units (A.U.). Results are representative of four experiments. Data are shown as mean (±SD). Value significantly different with * - control cells (without paraben and E2) (p ≤0.05); a – neutrophils incubated with paraben or E2 (p ≤0.05); b – neutrophils from women and men (p ≤0.05).

Article Snippet: 2,0, Latex Beads Polystyrene, Griess’ reagent for nitrite, Superoxide dismutase, N-Formylmethionineleucyl-phenylalanine, NBT Nitrotetrazolium Blue Chloride, Penicillin and Streptomycin antibiotic, Protease Inhibitor Cocktail (Merck Millipore, Burlington, USA), Anti-ERα (F-10): sc-8002 antibody, Anti-ERβ (B1): sc-390243 antibody, Anti-Neutrophil Elastase (G-2): sc-55549 antibody, Anti-Proteinase 3 (D-1): sc-74534 antibody, Anti-β-Actin (9): sc130301 antibody, ERβ receptor antagonist - PHTPP (Santa Cruz Biotechnology, Dallas, USA), GPER receptor antagonist - G-15 (Tocris Bioscience, Bristol, UK), Horse Anti-Mouse IgG secondary antibody (Vector Laboratories, Inc., Burlingame, USA), Mouse Anti-Goat IgG secondary antibody (Jackson ImmunoResearch, Cambridgeshire, UK), Vybrant® MTT Cell Proliferation Assay Kit, QubitTM Protein Assay Kit, Hoechst 33342 Fluorescent Nucleic Acid Stain, Myeloperoxidase Monoclonal Antibody (8E6)-FITC (Invitrogen, Waltham, USA), Annexin V-APC, BDTM Sheath Additive, 7-AAD (BD Biosciences, Jersey, USA), May-Grunwald and Giemsa solutions (Aqua Med, Łódź, Poland), RPMI1640 with L-glutamine and NaHCO3 (Biomed-Lublin, Lublin, Poland), PolymorphprepTM (Axis-Shield, Dundee, UK), MACS Separation Columns LS, CD16 MicroBeads Human and AutoMACS Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), Filters for Boyden Chamber, Boyden Chamber (Neuro Probe, Inc., Gaithersburg, USA), 3- Color Prestained Protein Marker (DNA Gdansk, Gdansk, Poland), Vacuette NH Sodium Heparin (Greiner Bio-One GmbH, Kremsmünster, Austria), 4 × Laemmli Sample Buffer, 2-mercaptoethanol, Mini TransBlot® Filter Paper, Ammonium Persulfate (APS), 10x Tris/Glycine Buffer, 10x Tris/Glycine/SDS, Stacking Gel Buffer for PAGE 0.5 M TrisHCl, pH 6.8, Resolving Gel Buffer for PAGE, Tween® 20, 1xTBS 1% Casein Blocker (Bio-Rad Laboratories, Inc., Hercules, USA), FSC (PANBiotech GmbH, Aidenbach, Germany), Trypan blue solution (Lachema, Praga, Czech Republic).

Techniques: Expressing, Western Blot, Incubation, Software, Control