Journal: Cell reports

Article Title: Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence

doi: 10.1016/j.celrep.2022.111756

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were then processed with TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Cat. No. SP-8400) to reduce the background following the manufacturer’s instruction and mounted in Anti-fade Mounting Medium with DAPI (Vector Laboratories, Cat. No. H-1800).

Techniques: Cell Culture, Produced, Recombinant, Infection, Western Blot, Lysis, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Blocking Assay, Stripping, Magnetic Beads, SYBR Green Assay, Luciferase, Amplification, Sequencing, shRNA, Software, Real-time Polymerase Chain Reaction

Journal: Crohn's & Colitis 360

Article Title: Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease

doi: 10.1093/crocol/otae003

Figure Lengend Snippet: (A) Overall in silico analysis of FPR1 in colonic pinch biopsies using Gene Expression Omnibus (GEO) GSE11223 and GSE20881 comparing IBD ( n = 207 colonic pinch biopsies; comprising UC and CD [ n = 124 and 83, respectively]) versus non-IBD controls ( n = 67; P = .0018). (B) FPR1 gene expression in UC noninflamed versus inflamed pinch biopsies ( n = 57 and 67, respectively), and CD noninflamed versus inflamed pinched biopsies ( n = 41 and 42, respectively; both P < .0001) Mann–Whitney test. FPR1 gene expression expressed as relative units to Stratagene Universal Human Reference Manual: Universal Human Reference RNA ( chem-agilent.com ). (C) Representative immunohistochemistry sections of inflamed and noninflamed colonic sections of IBD ( n = 17 CD and 24 CD, respectively), FPR1 is marked by horse-radish peroxide red (HRP) and neutrophils, elastase (DAB stained). (D) Quantification of FPR1+ve cells in CD and UC—average count/mm 2 of colonic section. Mann–Whitney statistics. ** P = .0002, *** P < .0001. (E) Representative immunofluorescence of UC and CD colonic sections—DAPI, FPR1, neutrophil elastase, and merged images. CD, Crohn’s disease; DAB, 3,3-diaminobenzidine tetrahydrochloride; DAPI, 4ʹ,6-diamidino-2-phenylindole; FPR1, formylated peptide receptor-1; IBD, inflammatory bowel disease; UC, ulcerative colitis.

Article Snippet: Sections were thoroughly washed and mounted using VECTASHIELD anti-fade mounting media with 4ʹ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and stored in the dark at 4°C until analyzed.

Techniques: In Silico, Expressing, MANN-WHITNEY, Immunohistochemistry, Staining, Immunofluorescence

Journal: Crohn's & Colitis 360

Article Title: Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease

doi: 10.1093/crocol/otae003

Figure Lengend Snippet: (A) Neutrophils were isolated from healthy human peripheral blood and separated using density gradient centrifugation. Purified neutrophils were labeled for multicolor flow cytometry. Expression of DAPI (dead cells), CD45 (general leukocytes), CD16 (neutrophils), CD11b (activated neutrophils), CD62L (primed neutrophils), and CD63 (activated neutrophils) were analyzed and a representative gating strategy was applied to identify activated neutrophils. (B) Quantification of CD11b+ neutrophils. (C) Quantification of CD63+/CD62L– neutrophils. (D) Percentage of activated CD11b+/CD62L–/CD63+ neutrophils in response to fMLF/synthetic ND6 stimulation and/or cyclosporin H (CsH) treatment. (E) Number of migrated neutrophils in response to fMLF/synthetic ND6 stimulation and/or CsH treatment. (F) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation. (G) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation with CsH treatment. (h) Extracellular neutrophil ROS production (with HRP to detect extracellular ROS) in response to fMLF/synthetic ND6 stimulation and CsH treatment (Figure H is Figure F and G combined). Data are means ± standard error (SEM) from n = 3 experiments performed in triplicate. Two-tailed t -test, Mann–Whitney, and 1-way ANOVA with Bonferroni correction and Dunnet’s tests were considered significant if P < .05 with an asterisk (*) indicating P < .05, double asterisks (**) indicating P < .001, and triple asterisks (***) indicating *** P < .0001. ANOVA, analysis of variance; DAPI, 4ʹ,6-diamidino-2-phenylindole; FPR1, formylated peptide receptor-1; HRP, horse-radish peroxide red; IBD, inflammatory bowel disease; UC, ulcerative colitis.

Article Snippet: Sections were thoroughly washed and mounted using VECTASHIELD anti-fade mounting media with 4ʹ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and stored in the dark at 4°C until analyzed.

Techniques: Isolation, Gradient Centrifugation, Purification, Labeling, Flow Cytometry, Expressing, Two Tailed Test, MANN-WHITNEY, IF-P

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

doi: 10.1007/s00018-013-1342-0

Figure Lengend Snippet: The secretion of intracellularly localized polySia-NCAM of human lung epithelial cell line A549 is induced by IL-1β and LPS. a Western-blot analyses of cell lysates of human A549 cells were performed using a mAb against polySia and NCAM (123C3) before and after endoN treatment. b, c Cultured A549 cells were fixed with methanol and stained with fluorescently labeled inactive endoN and b anti-NCAM mAb 123C3 or c polyclonal antibodies to visualize the trans-Golgi apparatus (anti-TGN38). For nuclear staining DAPI was used. d In addition, polySia was visualized in cells after stimulation with IL-1β (200 ng/ml) or LPS (100 ng/ml). Scale bars for c and d = 25 μm. e, f Polysialylated proteins were purified from supernatants of differently treated A549 cells and visualized by Western blotting using an anti-polySia mAb and after depolysialylation with a mAb against NCAM (123C3). g In an analogous cell lysates of IL-1β-treated cells were analyzed. h Western blots against polySia and NCAM (after depolysialylation) were performed using supernatants of untreated A549 cells as well LPS-stimulated cells in the absence or presence of TAPI-2. Apparent molecular masses of standard proteins are indicated in kDa

Article Snippet: For immunostaining against polySia, NCAM, and TGN38, cells were fixed with methanol at −20 °C for 10 min, blocked with 1 % BSA in PBS for 15 min, and incubation with appropriately diluted primary antibodies or biotinylated inactive endoN was continued for 1 h. After three washing steps with PBS and exposure to streptavidin–FITC conjugate and an anti-mouse-Rhodamine secondary antibody (Dianova, Hamburg, Germany), the final mounting VECTASHIELD with DAPI (Linaris, Munich, Germany) was added to the fixed cells.

Techniques: Western Blot, Cell Culture, Staining, Labeling, Purification