Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: MPO activity level in pancreas ( A ) and lungs ( B ) of healthy mice, mice with evoked acute pancreatitis (AP), and mice with AP after administration of 100% GBBC and FFAR1 or FFAR4 antagonist. Data presented as raw data (circles) and means (horizontal bolded lines) with SEM (boxes). Significance estimated with the use of one-way ANOVA followed by Dunnett’s post hoc test. # (hash) denotes comparison to control. * (asterisk) indicates comparison to AP. n = 4–8; * denotes p < 0.05; ** denotes p < 0.01; ###/*** denotes p < 0.001.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activity Assay, Comparison, Control

Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: Microphotographs of the pancreas. H&E staining: control group—normal architecture of the exocrine pancreas with functional acinar cells rich in zymogen granules ( A ); acute pancreatitis (AP) group—diffuse inflammatory infiltrates in periacinar area with the predominance of neutrophils ( B ); AP + 100% ghee butter from bovine colostrum (GBBC) group—acinar cell vacuolization, minimal atrophy of acinar cells with no evidence of inflammatory cell infiltrates ( C ); AP + FFAR1 antagonist + 100% GBBC group—dissociation and moderate atrophy of acinar cells ( D ); AP + FFAR4 antagonist + 100% GBBC group—dissociation of acinar cells, strong remodeling of the stroma, and marked atrophy of acinar cells ( E ). Masson’s trichrome staining: control group—minimal collagen deposition as a delicate framework for acinar cells ( F ); AP group—minimal collagen deposition in inflamed and edematous stroma ( G ); AP + 100% GBBC group—minimal collagen deposition between well-compacted acinar cells ( H ); AP + FFAR1 antagonist + 100% GBBC group—moderate collagen deposition in periacinar area and between individual acinar cells ( I ); AP + FFAR4 antagonist + 100% GBBC group—marked deposition of collagen between atrophied acinar cells ( J ). Immunoreactivity of α-SMA protein: control group—negative reaction ( K ); AP group—positive reaction in individual myofibroblast-like cells ( L ); AP + 100% GBBC group—moderate positive cytoplasmic reaction in migrating myofibroblast-like spindle-shaped cells penetrating periacinar spaces ( M ); AP + FFAR1 antagonist + 100% GBBC group—strong immunohistochemical reaction in myofibroblast-like cells ( N ); AP + FFAR4 antagonist + 100% GBBC group—strong immunohistochemical reaction in numerous myofibroblast-like cells ( O ). Nikon’s Eclipse E600 light microscope (Nikon Instruments Inc., Tokyo, Japan) was used to examine tissue samples. Microphotographs were taken with a digital imaging system that included a microscopy digital camera (Nikon DS-Fil, Nikon Instruments Inc., Tokyo, Japan) and image analysis software (NIS-Elements BR-2.20, Laboratory Imaging, Praha, Czech Republic); 400× magnification.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Staining, Control, Functional Assay, Immunohistochemical staining, Light Microscopy, Imaging, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Neutrophils in the Spotlight—An Analysis of Neutrophil Function and Phenotype in ARDS

doi: 10.3390/ijms252312547

Figure Lengend Snippet: Overview of fluorescently labeled antibodies. Abbreviations: phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC).

Article Snippet: fMLP-Receptor , APC , APC Anti-human, fMLP receptor Antibody Clone: REA169 Miltenyl Biotec, Bergisch Gladbach, Deutschland.

Techniques: Labeling

Journal: British Journal of Pharmacology

Article Title: Annexin‐A1 deficiency exacerbates pathological remodelling of the mesenteric vasculature in insulin‐resistant, but not insulin‐deficient, mice

doi: 10.1111/bph.14927

Figure Lengend Snippet: Expression and localisation of the AnxA1/FPR system in insulin‐resistant and insulin‐deficient mice. mAnxA1 (a), mFpr1, mFpr2, and mMCP‐1 mRNA expression in mesenteric arteries of insulin‐resistant (InsRes, b) and insulin‐deficient (InsDef, c) male mice. Sham AnxA1 +/+ and AnxA1 −/− (n = 6); insulin‐deficient AnxA1 +/+ and AnxA1 −/− (n = 6); insulin‐resistant AnxA1 +/+ and AnxA1 −/− (n = 6) male mice. Localisation (representative image) and quantification of FPRs in mesenteric arteries (d, n = 5–7). Insert highlights a region within the blood vessel (magnification ×100). VSMCs, vascular smooth muscle cells; EC, endothelial cell; mFpr1, formyl peptide receptor 1 gene; mFpr2, formyl peptide receptor 2 gene; FPR1, formyl peptide receptor 1 protein; FPR2, formyl peptide receptor 2 protein. Values are mean ± SEM. An outlier in insulin‐deficient AnxA1 −/− mice is predefined when an individual data point is 2 SDs away from the mean. *P<0.05, significantly different as indicated. (a) was analysed using unpaired Student's t test, (b) and (c) were analysed using repeated‐measures two‐way ANOVA with Bonferroni post hoc analysis

Article Snippet: To detect the expression of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=222&familyId=23&familyType=GPCR and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=223&familyId=23&familyType=GPCR in the vessel, sections were then incubated overnight at 4°C with either FPR1 primary antibody (1:100 in 5% NGS and 0.01% PBS‐T, Biorbyt Cat# orb13410, RRID:AB_10752393, Cambridge, UK) or FPR2 primary antibody (1:100 in 5% NGS and 0.01% PBS‐T, sc‐66901, Bioss Cat# bs‐3654R, RRID:AB_10858084, Texas, USA).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1

doi: 10.1038/s41598-017-07131-w

Figure Lengend Snippet: Honokiol inhibits superoxide anion generation and elastase release in FPR1-activated human neutrophils. Human neutrophils were incubated with 0.1% DMSO (as control) or honokiol (H; 1, 3, and 10 μM) for 5 min, and then activated by ( A ) fMLF (30 nM), ( B ) fMMYALF (300 nM), or ( C ) PMA (10 nM) for another 10 min. When fMLF and fMMYALF were used as activators, cells were primed by pre-incubation for 3 min with cytochalasin B (CB). Superoxide anion production was measured by ferricytochrome c reduction and elastase release was measured spectrophotometrically at 405 nm. ( D ) Xanthine oxidase was incubated with 0.1% DMSO (as control) or honokiol for 3 min, and then xanthine (0.1 mM) was added for 10 min. Reduction of WST-1 by cell-free xanthine/xanthine oxidase was measured spectrophotometrically at 450 nm. Superoxide dismutase (SOD; 20 U/ml) was used as a positive control. ( E ) Human neutrophils were incubated with 0.1% DMSO (as control) or honokiol for 15 min. Cytotoxicity was measured by assessing LDH release in cell-free medium. Total LDH release was determined by lysing cells with 0.1% Triton X-100 for 30 min at 37 °C. All data are expressed as the mean ± S.E.M. ( n = 3–5). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with ( A ) fMLF, ( B ) fMMYALF, or ( D ) control (0.1% DMSO).

Article Snippet: HEK-293 were stably transfected with the pCMV6-AC vector containing the human FPR1 gene (NM_002029; OriGene, Rockville, MD) using X-treme GENE Hp DNA transfection reagent (Roche, Mannheim, Germany).

Techniques: Incubation, Control, Positive Control

Journal: Scientific Reports

Article Title: Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1

doi: 10.1038/s41598-017-07131-w

Figure Lengend Snippet: Honokiol blocks fNLFNYK binding to FPR1 in human neutrophils. Human neutrophils were incubated with 0.1% DMSO (as control), honokiol (H; 3, 10, and 30 μM), or fMLF (10 μM) for 10 min and then labelled with FPR1-specific fluorescent ligand fNLFNYK (2 nM) for 20 min. ( A ) Representative histograms showing typical fluorescence in the absence or presence of fNLFNYK with honokiol or fMLF. ( B ) Mean fluorescence intensity is shown as the mean ± S.E.M. ( n = 4). ** P < 0.01; *** P < 0.001 compared with fluorescent dye alone.

Article Snippet: HEK-293 were stably transfected with the pCMV6-AC vector containing the human FPR1 gene (NM_002029; OriGene, Rockville, MD) using X-treme GENE Hp DNA transfection reagent (Roche, Mannheim, Germany).

Techniques: Binding Assay, Incubation, Control, Fluorescence

Journal: Scientific Reports

Article Title: Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1

doi: 10.1038/s41598-017-07131-w

Figure Lengend Snippet: Honokiol blocks fNLFNYK binding to FPR1 in FPR1-expressed neutrophil-like cells. ( A ) Dibutyryl cAMP-differentiated THP-1 or ( B ) hFPR1-transfected HEK-293 cells were incubated with 0.1% DMSO (as control), honokiol (H; 3, 10, or 30 μM), or fMLF (10 μM) for 10 min and then labelled with fNLFNYK (2 nM) for 20 min. ( A , B ) Representative histograms showing typical fluorescence in the absence or presence of fNLFNYK with honokiol or fMLF. ( C and D ) Mean fluorescence intensities of ( A and B ) are shown as the mean ± S.E.M. ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001 compared with fluorescent dye alone.

Article Snippet: HEK-293 were stably transfected with the pCMV6-AC vector containing the human FPR1 gene (NM_002029; OriGene, Rockville, MD) using X-treme GENE Hp DNA transfection reagent (Roche, Mannheim, Germany).

Techniques: Binding Assay, Transfection, Incubation, Control, Fluorescence

Journal: Scientific Reports

Article Title: Honokiol suppresses formyl peptide-induced human neutrophil activation by blocking formyl peptide receptor 1

doi: 10.1038/s41598-017-07131-w

Figure Lengend Snippet: Honokiol does not alter FPR1 mRNA expression and FPR2 binding effect in human neutrophils. ( A ) Human neutrophils were treated with 0.1% DMSO (as control), honokiol (10 and 30 μM), or fMLF (10 μM) at 4 °C for 30 min. Total RNAs were isolated with Trizol reagent and FPR1 mRNA levels were analyzed by quantitative PCR. ( B ) Human neutrophils were incubated with 0.1% DMSO (as control), honokiol (H; 10 μM), or WRW4 (1 μM) for 10 min and then labelled with FPR2-specific fluorescent ligand MMK-1F (200 nM) for 15 min. Representative histograms showing typical fluorescence in the absence or presence of MMK-1F with honokiol or WRW4. ( C ) Mean fluorescence intensity of ( B ) is shown. All data are expressed as the mean ± S.E.M. ( n = 3 or 4). *** P < 0.001 compared with fluorescent dye alone.

Article Snippet: HEK-293 were stably transfected with the pCMV6-AC vector containing the human FPR1 gene (NM_002029; OriGene, Rockville, MD) using X-treme GENE Hp DNA transfection reagent (Roche, Mannheim, Germany).

Techniques: Expressing, Binding Assay, Control, Isolation, Real-time Polymerase Chain Reaction, Incubation, Fluorescence

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: Summary of all IBD Gene Expression Omnibus (GEO) microarray/gene expression databases accessed for FPR1 gene analysis.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Gene Expression, Microarray, Expressing, Control

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 blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: In Silico, Gene Expression, 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) FPR1 gene expression in inflamed and noninflamed UC tissue from Gene Expression Omnibus (GEO) dataset GSE59071. (B–E) FPR1 gene expressions from GEO datasets GSE206285, GSE73661, GSE16879, and GSE92415, respectively. The gene expression units are (log 2 ) normalized gene expression in Robust Microarray Analysis (RMA) normalized format. Statistics: Mann–Whitney test with false discovery rate (FDR) P -value correction. FPR1, formylated peptide receptor-1; UC, ulcerative colitis.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Gene Expression, Microarray, MANN-WHITNEY

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–E) FPR1 gene expression in responders and nonresponders to (A) ustekinumab GSE206285; (B, C) infliximab GSE16879 and GSE23596, respectively; (D) vedolizumab GSE73661; and (E) golimumab GSE92415. Statistics: Mann–Whitney test and gene expression units are (log 2 ) normalized gene expression in Robust Microarray Analysis (RMA) normalized format. Statistics: Mann–Whitney test with false discovery rate (FDR) P -value correction. FPR1, formylated peptide receptor-1; IBD, inflammatory bowel disease; UC, ulcerative colitis.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Gene Expression, MANN-WHITNEY, Microarray

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: Difference in log 2 FPR1 expression in IBD, data available under Gene Expression Omnibus (GEO) databases above accessed for FPR1 gene analysis.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Expressing, Gene Expression

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) Percentage of weight loss in FPR1 –/– and wild-type C57/BL6 in 2% dextran-sulfate sodium (DSS) in drinking water ad libitum. (B) Representative H&E cross-section of distal colon in FPR1 –/– and wild-type following 7 days of DSS colitis. Bar is 500 µM. (C) Percent of inflamed distal colonic mucosa (ulcerated and loss of colonic epithelium/preserved noninflamed colonic epithelium with preserved crypt architecture) in FPR1 –/– and wild type following 7 days of DSS colitis. (D) Colon length in FPR1 –/– and wild type following 7 days of DSS colitis. (E) Quantification of LyG6+ve cells in the distal colon of FPR1 –/– and wild type following 7 days of DSS colitis—average count/mm 2 of colonic section. (F) Representative immunohistochemistry sections of distal colonic lamina propria of FPR1 –/– and wild type following 7 days of DSS colitis neutrophils Ly6G (DAB stained). FPR1, formylated peptide receptor-1; IBD, inflammatory bowel disease.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Immunohistochemistry, Staining

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 blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

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

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) Stool ND6 ELISA in IBD patients with active vs. highly active disease ( n = 16 and 27, respectively). (B) Stool ND6 ELISA in IBD patients with active disease, in remission and non-IBD subjects ( n = 45, 9, and 5, respectively). (C) Stool calprotectin s100a8/9 in IBD patients with active disease, in remission and non-IBD subjects ( n = 45, 9, and 6, respectively). (D) Stool ND6 ELISA in IBD patients with active disease stratified according to C-reactive protein < or >10 mg/L ( n = 22 and 21, respectively). (E) Correlation analyses of paired stool ND6 and blood C-reactive protein levels; 42 paired measurements. (F) Correlation analyses of paired stool ND6 and calprotectin s100a8/9 levels; 53 paired measurements. Data presented as mean ± standard error of the mean (SEM). Mann–Whitney statistical test between groups. Spearman correlation paired analyses. Significance level P < .05; ** P = .0008; *** P = .0007. FPR1, formylated peptide receptor-1; IBD, inflammatory bowel disease; NS, not significant.

Article Snippet: Sections were blocked in 2% fetal calf serum (FCS) diluted in TBS and incubated overnight in neutrophil elastase antibody (Novus Biologicals) and FPR1 antibody (Sigma-Aldrich) at 1:1000 and 1:500, respectively, at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Y. pestis AM18 (Δ yfeAB , Δ pgm ) and its variants POO1 ( yopE-dtx ), POO2 (Δ lcrV, yopE-dtx ) and POO3 (Δ lcrV (p lcrV ) , yopE-dtx ) were grown at 37°C with 5 mM EGTA to induce T3SS. Cultures were centrifuged to separate the supernatant (S) from the bacterial pellet (P) and extracts analyzed by immunoblotting with antibodies specific for YopE (αYopE), LcrV (αLcrV) and cytoplasmic RNA polymerase subunit A (αRpoA). b , Y. pestis cells (AM18, POO1, POO2 or POO3) were added at MOI of 10 to U937 for 4 hours at 37°C. Cell lysis was measured as LDH activity in centrifuged supernatants. SDS was used to generate a control sample. c , CRISPR-Cas9 mutagenesis of U937 cells was performed to select for variants resistant to Y. pestis POO1 intoxication as compared to Y. pestis POO2 control. Candidate genes were identified by next generation sequencing and data which are representative of three independent replicates were analyzed using the MaGeCK-based robust rank aggregation (RRA) score analysis. d , Y. pestis POO1 induced cell lysis in U937, FPR1 −/− and FPR1 −/− (pFPR1) cultures. e , Y. pestis KIM D27 (pMM83) mediated YopM-Bla translocation into U937, FPR1 −/− and FPR1 −/− (pFPR1) cells. Error bars represent the s.e.m. (n = 3 biological replicates) ( b , d , e ). One-way ANOVA with Bonferroni Correction was used to identify significant differences: ***, P <0.001; ns= not significant. One of three repeats is shown ( a - e ).

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Western Blot, Lysis, Activity Assay, Control, CRISPR, Mutagenesis, Next-Generation Sequencing, Translocation Assay

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Sequencing results for alleles cloned from CRISPR-Cas9 derived FPR1 −/− U937 macrophages. b , Immunoblot analysis for the production of FPR1, FPR2 and FPR3 in U937 macrophages, CRISPR-Cas9 derived FPR1 −/− cells and FPR1 −/− cells transfected with pFPR1. Numbers to the left of blots indicate migration of molecular weight markers. One of three repeats is shown ( a , b ).

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Sequencing, Clone Assay, CRISPR, Derivative Assay, Western Blot, Transfection, Migration, Molecular Weight

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Sequencing results for the alleles of two CCR5 −/− U937 isolates obtained using CRISPR-Cas9 mutagenesis. b , Cell survival following incubation with strains Y. pestis KIM D27 and POO1 was measured using the Trypan blue exclusion assay; error bars represent the s.e.m. (n = 4 biological replicates). T3SS injection into U937, FPR1 −/− , and CCR5 −/− by Y. pestis KIM D27 ( c ) and Y. pseudotuberculosis YPIII carrying pMM83 (YopM-Bla) ( d ); error bars represent the s.e.m. (n = 3 biological replicates). One of three repeats is shown ( b-d ). Statistical analysis was performed using one-way ANOVA with Bonferroni Correction: ***, P <0.001; **, P <0.01.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Sequencing, CRISPR, Mutagenesis, Incubation, Trypan Blue Exclusion Assay, Injection

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: Human neutrophils were stained with the β-lactamase substrate CCF2-AM, infected with wild-type Y. pestis KIM D27 (WT, pMM83) or KLD29 (Δ lcrV , pMM83) variant defective for the type III secretion system (T3SS), and analyzed for ( a ) blue fluorescence (YopM-Bla translocation into neutrophils and CCF2-AM cleavage) or ( b ) green fluorescence to derive the percent of stained cells injected with T3SS effector. c , Inhibition of Y. pestis T3SS into human neutrophils by monoclonal (αFPR1m) and polyclonal antibodies against FPR1 (αFPR1p), bacterial LcrV, N-formylpeptide (fMLF), annexin A1 peptide, staphylococcal CHIPS and cyclosporin H. d , Differential interference contrast (DIC) and fluorescence microscopy of mock or fMLF treated differentiated U937 cells infected with green-fluorescent Y. pestis KIM D27 (pEGFP) and stained with F1-specific antibody (red) to reveal extracellular bacteria in merged images of mock, but not in merged images of fMLF-treated cells. Orange and green arrows point to extra- and intracellular bacteria, respectively. e , Antibodies and ligands of FPR1 inhibit Y. pestis T3SS of YopM-Bla into U937 macrophages. One of three repeats is shown ( a-e ). Error bars represent the s.e.m. (n = 3 biological replicates) ( c , e ). One-way ANOVA with Bonferroni Correction was used to identify significant differences ( c , e ): ***, P <0.001; **, P <0.01.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Staining, Infection, Variant Assay, Fluorescence, Translocation Assay, Injection, Inhibition, Microscopy, Bacteria

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: Numbers of migrating immune cells were quantified in a transwell assay primed with mock, Y. pestis KIM D27 (WT) or KLD29 (Δ lcrV ) (10 7 CFU/ml). Chemotaxis toward fMLF is shown as a control: U937, FPR1 −/− and FPR1 −/− (pFPR1) cells ( a ); human neutrophils ( b ); granulocytes from wild-type (C57BL/6) and mFpr1 −/− mice ( c ). One of three repeats is shown and error bars represent the s.e.m. (n = 3 biological replicates). One-way ANOVA with Bonferroni Correction ( a , b ) and un-paired Student’s t -test ( c ) were used to identify significant differences: ***, P <0.001; **, P <0.01; *, P <0.05; ns, not significant.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Transwell Assay, Chemotaxis Assay, Control

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Numbers of migrating immune cells were quantified in a transwell assay primed with mock, 10 nM fMLF, 10 nM LTB4 or 100 ng ml −1 KC (CXCL1) for U937, FPR1 −/− and FPR1 −/− (pFPR1) cells. b , Numbers of migrating HL-60 cells were quantified in a transwell assay primed with mock, Y. pestis KIM D27 (WT) or KLD29 (Δ lcrV ) (10 7 CFU/ml). Chemotaxis toward fMLF is shown as a control. c , Increasing concentrations of LcrV S228 (10 −1 –10 3 ng/ml) were added to the transwell assay and number of migrating HL-60 cells recorded. Error bars represent the s.e.m. (n = 3 biological replicates); one-way ANOVA with Bonferroni Correction was used to identify significant differences: ***, P <0.001; **, P <0.01; *, P <0.05; ns, not significant. A representative of three independent experiments is shown.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Transwell Assay, Chemotaxis Assay, Control

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Wild-type Y. pestis KIM D27 or its Δ lcrV variant (KLD29) were added to U937 cells (MOI of 10) and adherence quantified as percent inoculum. b , Adherence of Y. pestis KIM D27 to U937, FPR1 −/− and FPR1 −/− (pFPR1) cells. c , Treatment of U937 cells with 10 μM fMLF or αLcrV or LcrV S228 (10–10 3 ng/ml) reduces Y. pestis KIM D27 adherence. One of three repeats is shown and error bars represent the s.e.m. (n = 3 biological replicates) ( a - c ). Significant differences were measured with the two tailed t -test ( a ), and one-way ANOVA with Bonferroni post-hoc analysis ( b , c ): ***, P<0.001; ns= not significant. d , Cleared detergent lysates of U937 or FPR1 −/− (pFPR1 STREP ) cells were incubated without (left panel) or with 10 MOI Y. pestis KIM D27 (right panel) and subjected to affinity chromatography on StrepTactin-sepharose. Load (L) and eluate (E) samples were analyzed by immunoblotting with IgG specific for FPR1, actin, YopD, LcrV, and RpoA. One of three repeats is shown.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Variant Assay, Two Tailed Test, Incubation, Affinity Chromatography, Western Blot

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Y. pestis KIM 8 and its variant KIM 8 Δ1234 (Yop-less) were grown at 37°C in TMH to induce T3SS. Cultures were centrifuged to separate the supernatant (S) from the bacterial pellet (P) and extracts were analyzed by immunoblotting with antibodies specific for YopB (αYopB), YopD (αYopD), YopE (αYopE), LcrV (αLcrV), YscF (αYscF), YopH (αYopH), YopM (αYopM) and YopJ (αYopJ). One of three repeats is shown. b , Wild-type Y. pestis KIM 8 or its Yop-less variant, KIM8 Δ1234, were added to U937 and FPR1 −/− cells (MOI of 10) and adherence quantified as percent inoculum. Error bars represent the s.e.m. (n = 3 biological replicates). One-way ANOVA with Bonferroni Correction was used to identify significant differences: **, P <0.01. A representative of three independent experiments is shown. c-d , Genetic complementation in FPR1 −/− cells using pFPR1 STREP . c , Y. pestis POO1 ( yopE-dtx ) but not POO2 (Δ lcrV , yopE-dtx ) induced cell lysis is restored in FPR1 −/− cells transfected with plasmid expressing C-terminal Strep-II tag FPR1 (pFPR1 STREP ). d , Y. pestis KIM D27 (pMM83) mediated YopM-Bla translocation into U937, FPR1 −/− and FPR1 −/− (pFPR1 STREP ) cells. One of three repeats is shown and error bars represent the s.e.m. (n = 3 biological replicates) ( c , d ). Significant differences were measured with one-way ANOVA with Bonferroni post-hoc analysis: ***, P <0.001.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Variant Assay, Western Blot, Lysis, Transfection, Plasmid Preparation, Expressing, Translocation Assay

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Transfection with p mFpr1 , but not p mFpr2 or p mFpr3 , restores Y. pestis KIM D27 (pMM83) translocation of YopM-Bla into U937 FPR1 −/− cells. One of three repeats is shown and error bars represent the s.e.m. (n = 3 biological replicates). b , Compared to C57BL/6 mice, BMDM from mFpr1 −/− , but not mFpr2 −/− mice, exhibit increased survival when infected with Y. pestis POO1 ( yopE-dtx ); one of three repeats is shown and error bars represent the s.e.m (n = 3 biological replicates). Y. pestis KIM D27 (pMM83) translocation of YopM-Bla in mouse BMDM ( c ) and neutrophils ( d ) requires mFpr1; one of three repeats is shown and error bars represent the s.e.m. (n = 3 biological replicates). e , Survival of wild-type C57BL/6 and mFpr1 −/− mice (n=10 males and 10 females) following subcutaneous inoculation with (600 CFU) Y. pestis CO92. f , Bacterial loads (CFU) in spleen tissues of dead and surviving animals. Horizontal bars denote the mean and error bars represent the s.e.m. One of two repeats is shown ( e , f ). Significant differences were determined using one-way ANOVA and Bonferroni’s post-hoc analysis ( a - d ) and the log-rank (Mantel-Cox) test ( e ): ***, P <0.001.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Transfection, Translocation Assay, Infection

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Quantification of Y. pestis KIM D27 (pMM83) translocation of YopM-Bla into neutrophils from five different donors (1–5) as compared to U937 cells. b , Quantification of migrating neutrophils from donors 2 and 4 following addition of fMLF (1–100 nM) or priming with mock, Y. pestis KIM D27 (WT) or KLD29 (Δ lcrV ) (2×10 7 CFU/ml). c , Model illustrating the position of amino acid substitutions in human FPR1. d , Surface display of FPR1 revealed by immunofluorescence microscopy using Alexa 488 labeled anti-FPR1 antibodies (αFPR1) ( left ). DAPI staining showing nuclei of U937 cells and variants ( right ). One of three repeats is shown. e , Quantification of migrating U937, FPR1 −/− , FPR1 −/− (pFPR1) and FPR1 −/− (pFPR1 R190W) cells in a transwell assay primed with mock, fMLF, Y. pestis KIM D27 (WT) or KLD29 (Δ lcrV ). One of three repeats is shown ( a , b , d , e ). Error bars represent the s.e.m. (n = 3 biological replicates) ( a , b , e ). One-way ANOVA and Bonferroni’s post-hoc analyses ( a , e ) and two-tailed t -test ( b ) were used to identify significant differences: ***, P <0.001; ns, not significant.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Translocation Assay, Immunofluorescence, Microscopy, Labeling, Staining, Transwell Assay, Two Tailed Test

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Serum derived from the blood of naïve or Y. pestis infected non-human primates (NHP, Cynomolgous macaque ) or human blood donors described in (donors 1–5) was analyzed for IgG specific for F1 antigen (αF1) via ELISA. b , List of amino acids changes deduced following cloning and sequencing of FPR1 and CCR5 alleles from human blood neutrophils (donors 1–5). c , Quantification of Y. pestis KIM D27 (pMM83) translocation of YopM-Bla into U937 or FPR1 −/− macrophages transfected with plasmids pFPR1 and pFPR1 R190W. d , Immunoblot analysis for the production of FPR1, FPR2, FPR3 and actin in U937 macrophages, and derived FPR1 −/− cells un-transfected or transfected with pFPR1 and pFPR1 R190W, respectively. One of three repeats is shown ( a , c ). Error bars represent the s.e.m. (n = 3 biological replicates) ( a , c ). One-way ANOVA and Bonferroni’s post-hoc analyses ( c ) was used to identify significant differences: ***, P <0.001.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Cloning, Sequencing, Translocation Assay, Transfection, Western Blot

Journal: Nature

Article Title: FPR1 is the plague receptor on host immune cells

doi: 10.1038/s41586-019-1570-z

Figure Lengend Snippet: a , Y. pestis releases N -formylpeptides via its type III secretion system (T3SS) in order to attract human neutrophils by activating N-formylpeptide receptor (FPR1) signaling and chemotaxis. b , The Y. pestis T3SS docks on the plague receptor (FPR1) via the LcrV needle cap protein. c , Docking promotes assembly of the membrane translocon (including LcrV, YopD and YopB), which provides a conduit for low-calcium signaling to the bacterial T3SS. d , Low-calcium signaling activates T3SS transport of Yop effectors into the cytoplasm, thereby killing host immune cells. e , LcrV shares homology with the annexin A1 peptide. Alignment performed using Clustal Omega.

Article Snippet: Plasmid encoding FPR1 ( FPR1 sgRNA resistant allele), which expresses human FPR1 from the SRα promoter was obtained from Addgene (plasmid #62600).

Techniques: Chemotaxis Assay, Membrane

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Tumor tissues were collected 14 days after inoculation with RM-1 cells in mice. TILs and TINKs were isolated. TILs were cultured with recombinant proteins in vitro, and recombinant protein tags were detected via flow cytometry. TINKs were cultured with recombinant proteins in vitro, and binding proteins were identified by IP-MS. FC, flow cytometry. ( B ) Venn diagram showing proteins with IP-MS scores ≥ 100 in TINKs, PBNKs, and PCa cells. ( C ) Proportion of membrane proteins versus nonmembrane proteins among overlapping proteins from B . ( D ) Membrane protein profiles identified by IP-MS. ( E ) Co-IP demonstrating mutual binding between ANXA6 and CypA-His. IB, immunoblotting. ( F ) Protein docking prediction between ANXA6 and CypA proteins using AlphaFold3. ( G ) Western blot analysis of ANXA6 expression and phosphorylation levels in NK cells treated with CypA and/or CsA. ( H ) Co-IP analysis of ANXA6 and FPR1 interaction in CypA-treated NK cells. ( I ) Fpr1 fl/fl mice were crossed with the Ncr1-iCre transgenic mice to generate the NK cell–specific Fpr1 -KO mice, which are denoted as Ncr1-iCre + -Fpr1 fl/fl . ( J ) Tumor growth curves and tumor weights in Fpr1 fl/fl and Ncr1-iCre + -Fpr1 fl/fl (cKO) mice inoculated with RM-1 vehicle or CypA-OE cells, respectively; N = 5 per group. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Tumor weight data are presented as mean ± SEM and were analyzed by 1-way ANOVA.

Article Snippet: 6- to 8-week-old male WT C57BL/6J mice, FVB mice, and NOG mice were purchased from Charles River Co., Ltd. Ncr1-iCre mice and Fpr1 fl/fl mice were purchased from Cyagen Biosciences.

Techniques: Isolation, Cell Culture, Recombinant, In Vitro, Flow Cytometry, Binding Assay, Protein-Protein interactions, Membrane, Co-Immunoprecipitation Assay, Western Blot, Expressing, Phospho-proteomics, Transgenic Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: PARP inhibitors restore NK cell function via secretory crosstalk with tumor cells in prostate cancer

doi: 10.1172/JCI197157

Figure Lengend Snippet: ( A ) Tumor growth curves and weights in PARPi-treated tumor-bearing mice coadministered with CypA inhibitor CsA and FPR1 inhibitors CsH and HCH6-1; N = 6 per group; scale bars: 1 cm ( B ) Tumor growth curves and weights in C57BL/6 mice bearing RM-1 tumors treated with FPR1 agonist fMIFL or inhibitor HCH6-1; N = 5 per group; scale bars: 1 cm ( C and D ) IFN-γ and GZMB expression levels in TINKs ( C ) and their quantification ( D ) under HCH6-1 or fMIFL treatment; N = 5 per group. ( E ) Western blot analysis of ERK- and AKT-related pathway activation in NK cells treated with CypA, CsA, CsH, or HCH6-1 in vitro. ( F and G ) Flow cytometry analysis of IFN-γ and GZMB expression ( F ) and quantification ( G ) in NK cells treated with FPR1 inhibitor CsH, AKT inhibitor MK-2206, or ERK inhibitor SCH772984; N = 5 per group. ( H ) Transmission electron microscopy images showing mitochondrial structure and crista alterations in NK cells stimulated with CypA. Scale bars: 1 μm. ( I ) Statistical analysis of mitochondrial number ( N = 10) and crista count ( N = 15) in NK cells. Tumor growth curve data are presented as mean ± SD and were analyzed by 2-way ANOVA with Tukey’s multiple-comparison test. Other data are presented as mean ± SEM and were analyzed by 1-way ANOVA ( A , B , D , and G ) and Welch’s t test ( I ).

Article Snippet: 6- to 8-week-old male WT C57BL/6J mice, FVB mice, and NOG mice were purchased from Charles River Co., Ltd. Ncr1-iCre mice and Fpr1 fl/fl mice were purchased from Cyagen Biosciences.

Techniques: Expressing, Western Blot, Activation Assay, In Vitro, Flow Cytometry, Transmission Assay, Electron Microscopy, Comparison

Journal: The Journal of pathology

Article Title: ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

doi: 10.1002/path.6288

Figure Lengend Snippet: Figure 2. ARAP3 protects from formylated peptide-induced endo- thelial permeability. (A) Bone marrow chimeras as indicated were challenged with fMLF i.t. BALf was harvested 6 h later for analysis of total protein. (B–E) Monolayers of ARAP3-deficient b.End5 or HUVECs and matched controls were stimulated with fMLF or vehi- cle. (B and C) RTCA of electrical impedance measurements was performed to characterise b.End5 monolayer permeability. (B) Time course. (C) AUC. (D and E) Transwell FITC-dextran perme- ability of vehicle or fMLF-stimulated (D) bEnd5 and (E) HUVEC monolayers that had been transiently transfected with scrambled control or ARAP3 targeting siRNA. (A) Each symbol represents one experimental animal. Experiments performed on two separate occa- sions are pooled. (B–E). Graphs show mean ± SEM and combine results obtained in four separately conducted experiments. Transwell experiments are expressed as fMLF-stimulated experi- mental condition over its vehicle-stimulated control. Analysis was by two-way ANOVA with Tukey’s multiple comparison test or (B) unprotected Fisher’s least significant difference multiple compar- ison test. *, #p < 0.05; **, ##p < 0.01; ***p < 0.001, ****p < 0.0001; ns, not significant. Red asterisks in (B) refer to significant differences between fMLF-stimulated and vehicle-treated Arap3/ cells; red number symbols refer to significant differences between fMLF-stimulated Arap3/ and control cells.

Article Snippet: For siRNA-mediated knock-down, HUVECs were transfected with siRNA oligonucleotide duplexes targeting the gene of interest (ARAP3: SR312043; FPR1: SR301651) or control siRNA (SR30004) obtained from Origene (Herford, Germany).

Techniques: Permeability, Transfection, Control, Comparison

Journal: The Journal of pathology

Article Title: ARAP3 protects from excessive formylated peptide-induced microvascular leakage by acting on endothelial cells and neutrophils.

doi: 10.1002/path.6288

Figure Lengend Snippet: Figure 3. ARAP3 regulates endothelial FPR1 and VE-cadherin. HUVECs were transfected with scrambled (Ctrl) siRNA or siRNA targeting FPR1 or ARAP3 as indicated. (A–D) FPR1 was detected using flow cytometry. (A and C) Histogram of representative experiments with isotype control in black, control siRNA-transfected cells in grey and (A) FPR1 or (C) ARAP3 siRNA transfected cells in red. (B and D) Mean fluorescence intensity (MFI) of six separately conducted experiments. (E) Expression of FPR1 mRNA performed with three independent transfections. (F) Transwell FITC-dextran permeability combining a minimum of four separately conducted experiments. In the legend of this graph, ARAP3 is shortened to A3. (G–J) HUVECs transfected with siRNA as indicated were plated onto glass coverslips, allowed to form confluent monolayers, and stimulated with 100 nM fMLF. (G) Schematic diagram of antibody feeding experiments. (H) VE-cadherin analysis by confocal microscopy. (I) Automated analysis of co-localisation was performed with Huygens Software as detailed in Materials and methods. Co-localisation according to efficient i is plotted. Each symbol represents a separate raw image; images were obtained from a minimum of two separate transfections. (J) Fixed, permeabilised cells were labelled for VE-cadherin, LAMP-1, and Rab11A. (H and J) Examples of representative flattened confocal image stacks; scale bars, 10 μm (H) and 20 μm (J). Data were analysed by (B, D, and E) one-way ANOVA with Dunnet’s multiple comparison test, comparing all experimental conditions to control siRNA transfected cells, (F) two-way ANOVA with Tukey’s multiple comparison test with asterisk referring to activation and number symbol to differences between conditions tested and (I) unpaired two-tailed t-test. *, #p < 0.05; **p < 0.01.

Article Snippet: For siRNA-mediated knock-down, HUVECs were transfected with siRNA oligonucleotide duplexes targeting the gene of interest (ARAP3: SR312043; FPR1: SR301651) or control siRNA (SR30004) obtained from Origene (Herford, Germany).

Techniques: Transfection, Cytometry, Control, Expressing, Permeability, Confocal Microscopy, Software, Comparison, Activation Assay, Two Tailed Test