Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: TLR2 −/− , NOD2 −/− , FPR1 −/− and wt mice were inoculated intradermally with S. aureus . (A) Mean total lesion size (cm 2 ) ± SEM. (B) In vivo bioluminescence quantified by mean total flux (photons/s) ± SEM (logarithmic scale). (C) Mean IL-1β protein levels (pg/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. (D) Mean myeloperoxidase (MPO) activity (U/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. * p<0.05, † p<0.01, TLR2 −/− , NOD2 −/− or FPR1 −/− mice versus wt mice (Student's t -test). Data are from 2 experiments with at least 4 mice/group per experiment.

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: In Vivo, Activity Assay

Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: Neutrophils from mouse bone marrow were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. IL-1β protein levels (mean ± SEM) were measured in culture supernatants by ELISA. (A, B) IL-1β protein levels in supernatants from S. aureus -infected neutrophils from TLR2 −/− , NOD2 −/− , FPR1 −/− , ASC −/− and wt mice. (C, D) IL-1β protein levels in supernatants from wt mouse neutrophils infected with S. aureus (C) or MRSA (D), in the absence and presence of an NLRP3-inhibitor (glibenclamide), a caspase-1 inhibitor (Z-YVAD-FMK) or anti-staphylococcal α-toxin antiserum. The respective vehicle controls (DMSO or normal rabbit IgG) for the inhibitors had IL-1β protein levels that did not differ than media alone (data not shown). Data are from 3–5 mice per group. *p<0.05; † p<0.01, ‡ p<0.001, TLR2 −/− , NOD2 −/− , FPR1 −/− or ASC −/− mice versus wt mice (A, B) or the NLRP3-inhibitor, caspase-1 inhibitor or anti-staphylococcal α-toxin antiserum treatment versus media alone (C, D) (Student's t -test).

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Materials Today Bio

Article Title: Co-delivery of panobinostat and siSTAT3 using engineered M1 exosomes to establish a one-two punch therapeutic strategy for glioblastoma recurrence

doi: 10.1016/j.mtbio.2025.102680

Figure Lengend Snippet: Inflammation-directed targeted delivery in vitro. (A) Representative confocal images of the uptake of M1-EXOs and iM1-EXOs-PAN by GB cells. Scale bar, 25 μm. (B) Quantification of the PKH67‐positive cell ratio based on the confocal microscopy images. (C) Schematic illustration of the in vitro BBB model using a trans-well system to evaluate the penetration capability of iM1-EXOs-PAN across the endothelial monolayer. The transwell co-culture system containing HUVEC cells (simulating the vascular endothelial cells of BBB) in the upper chamber and GB cells in the bottom chamber. And, iM1-EXOs-PAN was added to the upper chamber, and FBS free medium with or without fMLP (100 μM, MCE, HY-P0224) was added to the lower chamber. (D) Representative images of HUVECs and LN229 cells up-taking the nanoformulation treated with chemotactic peptide (+fMLP, 100 μM) or none (-fMLP) at 12 h. Scale bar, 20 μm. (E) The percentage of PKH67 positive cells treated with chemotactic peptide (+fMLP, 100 μM) or none (-fMLP) at 4, 8, and 12 h. Statistical significance was calculated using two-way ANOVA. n. s = not significant, and ∗∗∗ P < 0.001.

Article Snippet: Monolayered HUVEC with TEER values greater than 300 Ω cm 2 were adopted as the BBB model. Then, 0.2 μg/μL of PKH67-labeled iM1-EXOs-PAN was added to the upper chamber, and FBS-free medium with or without fMLP (100 μΜ, HY-P0224, MCE) was added to the lower chamber [ ].

Techniques: In Vitro, Confocal Microscopy, Co-Culture Assay

Journal: The Journal of Neuroscience

Article Title: Resolvin D1 Programs Inflammation Resolution by Increasing TGF-β Expression Induced by Dying Cell Clearance in Experimental Autoimmune Neuritis

doi: 10.1523/JNEUROSCI.0020-16.2016

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: The following antibodies were used in this study: ED1 antibody for macrophages (1:100; Serotec), W3/13 antibody for T lymphocytes (1:50; Serotec), OX22 antibody for B cells (1:200; Serotec), ED2 antibody for anti-inflammatory macrophages (1:100; Serotec), or FPR2 antibody for ALX receptor (1:50; Alomone).

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: Resolvin D1 Programs Inflammation Resolution by Increasing TGF-β Expression Induced by Dying Cell Clearance in Experimental Autoimmune Neuritis

doi: 10.1523/JNEUROSCI.0020-16.2016

Figure Lengend Snippet: Primers used in this study

Article Snippet: The following antibodies were used in this study: ED1 antibody for macrophages (1:100; Serotec), W3/13 antibody for T lymphocytes (1:50; Serotec), OX22 antibody for B cells (1:200; Serotec), ED2 antibody for anti-inflammatory macrophages (1:100; Serotec), or FPR2 antibody for ALX receptor (1:50; Alomone).

Techniques:

Journal: Frontiers in Immunology

Article Title: Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients

doi: 10.3389/fimmu.2018.01968

Figure Lengend Snippet: G-CSF treatment affects neutrophil migration. Stem cell donors before and after treatment were analyzed for neutrophil function. (A) Neutrophil count measured before and after G-CSF-treatment in the stem cell donor group ( n = 10). Revealed a significant increase in the number of circulating neutrophils after G-CSF treatment. (B) Distribution of half migration time during chemotaxis within the stem cell donor group before and after G-CSF-treatment. Half migration time was defined as the time required for half-maximal fluorescence signal indicative for migration toward 10 nM fMLP. (C) The distribution of velocity during chemotaxis before and after G-CSF-treatment is indicated. Velocity was measured as Δ %/min at half migration time indicating the speed of the neutrophils when they have reached half maximal fluorescence in the bottom well. Blood cell differentiation analysis (D) before and (E) after purification of neutrophils from stem cell donors before ( n = 3) and after ( n = 4) GCSF treatment confirmed that the purification enriched mature neutrophils >95% independent of previous G-CSF treatment. Statistical analysis was performed using a Wilcoxon signed-rank test (A–C) . * p < 0.05, ** p < 0.01.

Article Snippet: G-CSF treated as well as untreated neutrophils were stained with labeled monoclonal anti-fMLP receptor antibody coupled to PE (PE, MACS, Miltenyi Biotec) in PBS supplemented with 3% FCS in a final dilution of 1:10 and incubated for 30 min at 8°C.

Techniques: Migration, Chemotaxis Assay, Fluorescence, Cell Differentiation, Purification

Journal: Frontiers in Immunology

Article Title: Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients

doi: 10.3389/fimmu.2018.01968

Figure Lengend Snippet: Systemic G-CSF treatment impairs chemotaxis in neutrophils from healthy donors. Purified neutrophils from peripheral blood of healthy, untreated donors ( n = 8) were incubated with 0, 10, or 50 ng/ml human recombinant G-CSF. Thereafter, a chemotaxis assay was performed using fMLP as chemoattractant and half migration time (A) as well as velocity at half migration time (B) was determined. Neutrophils pre-treated with 0, 10, and 50 ng/ml of G-CSF respectively were stained with a PE-labeled anti-fMLP receptor antibody. The histogram is showing a representative experiment of three similar replications (C) . Statistical analysis was performed with a Kruskal-Wallis test for (A) ( p = 0.0098) and (B) ( p = 0.0093), complemented with Mann-Whitney U-tests showing that samples 0 and 50 ng/ml are significantly different for (A,B) with * p < 0.05.

Article Snippet: G-CSF treated as well as untreated neutrophils were stained with labeled monoclonal anti-fMLP receptor antibody coupled to PE (PE, MACS, Miltenyi Biotec) in PBS supplemented with 3% FCS in a final dilution of 1:10 and incubated for 30 min at 8°C.

Techniques: Chemotaxis Assay, Purification, Incubation, Recombinant, Migration, Staining, Labeling, MANN-WHITNEY

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Model of allergen-induced airway hyperresponsiveness (AHR) for wild-type (WT) and Saa–/– mice (both C57BL/6 background). Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 1a-​-jj and Extended Data Fig. 2 and ​and3).3). (b) For SAA1 antibody blockade, we used an established mouse model of allergen-induced AHR sensitizing WT BALB/cJ mice i.t. on day 0 and 14 with 100 µg of HDM extract + isotype control, or HDM + αSAAab. Airway measurements and tissue harvests were performed 72 h after the last allergen challenge (used in Extended Data Fig. 4). (c) In short-term exposure protocols WT and Saa–/– mice (both C57BL/6 background) received a single HDM challenge (100 μg) were sacrificed 16 h later (used in Fig. 2a-​-d).d). (d) In short-term exposure protocols BALB/cJ mice received a single HDM challenge (100 μg)+ isotype control, HDM challenge + HDL (200μg) and isotype control, or HDM + αSAAab and were sacrificed 24 h later (used in Fig. 2e). Contol mice received either PBS + isotype or PBS + HDL and isotype control. (e) For overexpression of SAA1 in vivo, mice were injected 20 µg of DNA complexed to polyethylenimine at day 0, exposed to PBS or HDM 48 h later and ILC2s as well as BAL cytokines measurements were performed on day 3 (used Fig. 2f). (f) WT and Saa–/– mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 μg with extracts from the parasitic worm Schistosoma mansoni (a Puerto Rican isolate). Tissues were harvested 72 h after the last allergen challenge (used in Fig. 4). (g) For FPR2 blockade (WRW4, 2 mg/kg), WT BALB/cJ mice were sensitized and challenged i.t. on day 0 and 14 with 100 μg of HDM extract. Airway measurements were performed 72 h after the last allergen challenge (used in Fig. 7a-​-f).f). (h) In short term exposure experiments, BALB/cJ mice received a single HDM challenge (100 μg) or HDM + WRW4 and were sacrificed 24 h later (used in Fig. 7g and ​andi).i). (i) Model of Alternaria alternata (Alt a )-induced airway inflammation for WT and Saa–/– mice. Mice were sensitized i.t. on day 0 (1 μg) and i.n. on days 7–11 with 10 µg of Alt a extract. Tissues were harvested 72 h after the last allergen challenge (used in Extended Data Fig. 9f-​-jj).

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Over Expression, In Vivo, Injection

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 mRNA expression in nasal epithelial cells of CRS patients and matched control donors. (c) HDM (100 μg/ml)-triggered IL-33 concentration in primary nasal epithelial cells of CRS patients as compared to controls. (d) Dissociation of hexameric SAA was analyzed separating lipid-free and lipid-bound SAA by HDL pull down using a polyclonal goat antibody specific for human ApoA1 and immunoblotting with a monoclonal mouse antibody specific for human SAA1 (Acris). (e) Bar graph represents quantitative analysis of SAA monomer band intensities (LI-COR Image Studio Software). (f) SAA1 protein amounts in sera of control donors and HDM allergic individuals. Data represents means ± SEM of (a and b) n=16 control and n=27 CRS, (c) n=6 control and n=12 CRS patients per group, (e) n=5 control and n=8 CRS patients per group and (f) n=18 control and n=27 HDM allergic patients per group. Representative immunoblot of one control and one CRS patient (d). Cropped images are shown. P values were calculated with a two-tailed test using Student’s t-test with Welch’s correction. ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing, Concentration Assay, Western Blot, Software, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Airway responses to cholinergic stimulation shown as airway pressure over time (APTI) measured on day 17 in PBS, HDM, or HDM+WRW4-treated (4 mg/kg) WT BALB/c mice (**P = 0.0022). (b) Total IgE serum concentrations of treated mice (HDM to PBS **P = 0.0483; HDM to WRW4 **P = 0.0486). (c) Eosinophilic infiltration into the lungs (**P = 0.0094 and ***P = 0.0009). (d and e) Histological examination of airway inflammation. Sections were stained for mucus production with periodic acid Schiff (PAS; Scale bars 100 μm). (f) Lung expression of Il5 (*P = 0.0421 and ***P = 0.0029) and Il13 (***P = 0.0009) mRNA. (g) BAL IL-33 (*P = 0.0175 and **P = 0.0009) and (h) Frequency of Lin-CD45+ST2+IL-13+ ILC2s (*P = 0.0125 and **P = 0.0058) and (i) BAL IL-13 (HDM to PBS *P = 0.0169; HDM to WRW4 *P = 0.0398) in the lung 24 h after a single i.t. challenge with PBS, HDM (100 μg), or HDM in combination with the FPR2 inhibitory peptide WRW4 (4 mg/kg). Data represents means ± SEM of pooled data from 2 experiments containing (a, b, f) n=11 PBS, n=11 HDM, n=14 HDM+WRW4 animals per group; (d, e) n=2 PBS, n=3 HDM, n=3 HDM+WRW4 animals per group; (g) n=11 PBS, n=13 HDM, n=14 HDM+WRW4 animals per group; (h) n=15 PBS, n=15 HDM, n=15 HDM+WRW4 animals per group; or are representative of 2 (c) or 3 (i) independent experiments with (c) n=5 PBS, n=5 HDM, n=7 HDM+WRW4 animals per group and (i) n=8 animals in each group, respectively. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc (a-c, e, g-i) or Holm-Sidak’s (f) post hoc analysis that compares HDM to PBS and WRW4 counterparts. ****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Staining, Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) mRNA expression of the FPR family members FPR1, FPR2 and FPR3 at baseline (open bars) or after 2 h of HDM stimulation (filled bars). (b) HDM-triggered IL-33 amounts in BEAS-2B cells overexpressing human FPR1 (**P = 0.0068, ***P = 0.0003). (c) IL-33 secretion in BEAS-2B cells overexpressing human FPR2 or cells transfected with an empty vector (EV; pcDNA3.1) (**P = 0.0068, ***P = 0.0003). HDM-induced IL-6 and IL-8 amounts in BEAS-2B cells overexpressing FPR2 (d and e) or blocking the FPR2 receptor (f (**P = 0.0043) and g (**P = 0.0021)) using WRW4. Data presented as means ± SEM and is representative of 2 independent experiments each containing at least n= 4 biologically independent samples (b, d, f) or pooled data from 2 independent experiments (c, e, g). mRNA expression, normalized to the average of housekeeping genes, is presented as mean values ± SEM (n = 5 biologically independent samples) performed in duplicates (a). P values were calculated with a two-tailed test using Student’s t-test (a) or one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (b-e) or Dunett’s post hoc analysis (f, g). ****P ≤ 0.0001

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing, Transfection, Plasmid Preparation, Blocking Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: IL-33 concentrations in BEAS-2B cells blocking the SAA-binding receptors (a) FPR2 (WRW4, 12 μM; ***P = 0.0006) and (b) TLR4 (LPS from Rhodobacter sphaeroides; LPS-RS, 10 μg/ml; ). (c) Effects of FPR2 or TLR4 blockade in cells transfected with empty plasmid control (EV) or SAA1 overexpression plasmid. **P = 0.0047 § indicates p = 0.056; # indicates p = 0.22 between EV and SAA plasmid. Blockade of (d) CD36 (anti-human CD36 blocking antibody, aCD36; 10 μg/ml; ***P = 0.0005), (e) the P2 receptor antagonist suramin (100 μM; *P = 0.028), and (f) TLR2 (anti-human TLR2 IgA, aTLR2, 10 μg/ml; **P = 0.0055). (g) HDM-triggered IL-33 release in BEAS-2B cells transfected with EV, WT SAA1 overexpression plasmid or a SAA1 plasmid with a deletion of the C-terminal amino acids 1–11 (Δ1–11; *P = 0.01, **P = 0.0078 ). Data are representative of 2–3 independent experiments (b, d-f) or pooled data from 2 independent experiments (a, c, d, g) each containing at least n=4 biologically independent samples and depicted as means ± SEM. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s (a, b, d-g) or Tukey’s (c) post hoc analysis.****P ≤ 0.0001.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Blocking Assay, Binding Assay, Transfection, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: (a) Immunoblot of SAA1 after Alternaria alternata (Alt a) stimulation of BEAS-2B cells performed as described in Fig. 3b. Alt a-induced IL-33 and IL-8 (**P = 0.0044) secretion in BEAS-2B cells after siRNA-mediated silencing of SAA1 (siSAA1) (b and c) or WRW4-mediated FPR2 blockade (d and e). (f) Total serum IgE concentrations, (g) eosinophil counts and frequency of (h) CD3+CD4+, (i) TH2 and (j) TH17 cells in the lungs of PBS or Alt a-treated WT and Saa–/– mice. Immunoblots are representative of an experimental n=2 (a). Data are presented as means ± SEM and represent pooled data from 2 independent experiments (b, d, e, f, g, i, j) or show one representative experiment (c) each containing at least n= 4 biologically independent samples or n=8 WT PBS, n=13 WT Alt a, n=8 Saa–/– PBS and n=11 Saa–/– Alt a animals per group (f, g, i, j) or n=5 WT PBS, n=9 WT Alt a, n=5 Saa–/– PBS and n=7 Saa–/– Alt a animals per group (h). Cropped images are shown. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Tukeys multiple comparison test (a, b) or Dunett’s post hoc analysis (d-j). ****P ≤ 0.0001 siNT = non-targeting siRNA; siSAA1 = SAA1-targeting siRNA. ns=not significant.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Western Blot, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Basal (a) SAA1 and (b) FPR2 expression in bronchial epithelial from asthmatic patients and matched controls. Data represents means ± SEM of (a and b) n= 6 control and n= 6 asthmatic patients per group. x.

Article Snippet: Human FPR2 (#SC322591, Origene) and human SAA1 (#RC202738, Origene); murine rFPR1 (#MR221675, Origene), pcDNA3.1 (Invitrogen, Thermo Scientific). hFPR2, hSAA1, and On-TARGET plus Non-targeting siRNA (Smart pool: ON-TARGETplus siRNA, GE Dharmacon,); BEAS-2B and Caco-2 cell line was from American Type Culture Collection (ATCC).

Techniques: Expressing

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet:

Article Snippet: AGER antibody , Proteintech , Cat#Ag23105; RRID:Addgene_80089.

Techniques: Marker, Polymer, Plasmid Preparation, Recombinant, Protease Inhibitor, Saline, Enzyme-linked Immunosorbent Assay, Activation Assay, Silver Staining, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Transfection, Gene Expression, Reporter Assay, Staining, Over Expression, Software, Imaging, Live Cell Imaging, Microscopy

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Expression of FPR2 mRNA in epithelial ovarian cancer (EOC) and normal ovarian tissues. (A) The FPR2 mRNA expression level in EOC was significantly higher than that in the normal ovarian tissues (*P<0.05). (B) The FPR2 mRNA expression was significantly higher in the SKOV3 cell line than that in the other three ovarian cancer cell lines (**P<0.01).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Immunohistochemical staining pattern of FPR2 in EOC and control tissues. (A) FPR2 expression was not clearly observed in the normal ovarian tissues, and was scored as weak expression (+). (B) FPR2 was moderately expressed on the cytomembrane and cytoplasm of EOC cells (++). (C) FPR2 was strongly expressed on the cytomembrane and cytoplasm of EOC cells (+++).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Immunohistochemical staining, Staining, Control, Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Correlation between FPR2 mRNA expression and clinicopathological characteristics of the EOC cases.

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Correlation between FPR2 protein expression and clinicopathological characteristics of the EOC cases.

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Overall survival (OS) of EOC patients were analyzed using the Kaplan-Meier test. (A) Patients with high FPR2 expression showed a significantly lower OS compared with patients with low FPR2 expression (P<0.001). (B). In high-grade EOC patients, high FPR2 expression was associated with significantly lower OS compared with low FPR2 expression (P=0.014). (C) In low-grade EOC patients, high FPR2 expression showed no significant difference from low FPR2 expression in terms of the OS of EOC patients (P=0.077). (D) In advanced FIGO stages, high FPR2 expression indicated significantly lower OS compared with low FPR2 expression (P=0.007). (E) In early FIGO stages, high FPR2 expression showed no significant difference from low FPR2 expression in terms of the OS of EOC patients (P=0.422).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: Univariate and multivariate analyses of prognostic factors for EOC.

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: (A) Validation of FPR2 expression in FPR2-knockdown SKOV3 cells using RT-qPCR and Simple Western assays. The mRNA and protein levels of FPR2 were significantly decreased in the SKOV3 −shFPR2 cells compared with the control cells, with the SKOV3 −shFPR2-2 cells exhibiting the greatest inhibition. (B) The wound healing assay showed that FPR2 knockdown significantly decreased the cell migration rate at 4 h after scraping. Significantly different from the control (SKOV3 −shcontrol ; *P<0.05). (C) The Transwell assay showed that FPR2 knockdown decreased the number of invasive SKOV3 cells (*P<0.05).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Biomarker Discovery, Expressing, Knockdown, Quantitative RT-PCR, Simple Western, Control, Inhibition, Wound Healing Assay, Migration, Transwell Assay

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: (A) Compared with untreated cells, cells treated with one of three concentrations of SAA (0.1, 1 and 10 µg/ml) showed markedly decreased FPR2 mRNA expression (**P<0.01). (B) The wound healing assay showed that the average migration rate of SKOV3 cells was significantly increased after treatment with 0.1 µg/ml SAA, whereas the other concentrations showed no significant difference compared with the NC group (*P<0.05). (C) The Transwell assay showed that there was no significant difference in the number of cells that penetrated the Matrigel among the different groups (P>0.05). (D) The wound healing assay showed that the average migration rate of the SAA+SKOV3 −shFPR2 cells was significantly decreased compared with the NC group at both 2 and 4 h after scraping (**P<0.01, *P<0.05). (E) The Transwell assay showed that the number of invasive SAA+SKOV3 −shFPR2 cells was significantly decreased compared with the NC group (*P<0.05).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Expressing, Wound Healing Assay, Migration, Transwell Assay

Journal: Oncology Reports

Article Title: Formyl peptide receptor 2 expression predicts poor prognosis and promotes invasion and metastasis in epithelial ovarian cancer

doi: 10.3892/or.2017.6034

Figure Lengend Snippet: (A) Tube formation assay showed that FPR2 knockdown led to a significant reduction in tube length compared with the NC group (*P<0.01). The SAA+RNAi group showed a significant increase in tube length. Significantly different from the RNAi group ( # P<0.01). The SAA+NC group showed a significantly increase in tube lengths. Significantly different from the NC group ( △ P<0.01). (B) The Simple Western results showed that FPR2 knockdown decreased the VEGF protein expression compared with the NC group (*P<0.01). Compared with the RNAi group, the SAA+RNAi group presented a marginal increase in VEGF expression, whereas the difference between the two groups was not statistically significant (P>0.05). No significant difference in VEGF expression was found between the NC and SAA+NC group (P>0.05).

Article Snippet: The sections were incubated with the FPR2 antibody (13448–1-AP; 1:50 dilution; Proteintech, San Diego, CA, USA) at 4°C overnight and subsequently incubated with a streptavidin-peroxidase system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer's instructions.

Techniques: Tube Formation Assay, Knockdown, Simple Western, Expressing

Journal: PLoS ONE

Article Title: Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1

doi: 10.1371/journal.pone.0138338

Figure Lengend Snippet: Blocking of FPR1 abrogates neutrophil migration to fMet-Leu-Phe and gliadin. Pretreatment of neutrophils with cyclosporine H, a specific inhibitor of FPR1, completely abrogated the neutrophil migration induced by PT-gliadin and fMet-Leu-Phe. As expected, LTB 4 induced neutrophil chemotaxis was not affected by cyclosporine H.

Article Snippet: HEK293T cells were transfected with human FPR1 according to the manufacturer’s protocol (Origene cDNA ORF Clone, OriGene Technologies, Rockville, MD).

Techniques: Blocking Assay, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1

doi: 10.1371/journal.pone.0138338

Figure Lengend Snippet: Blocking of FPR1 inhibits neutrophil migration to gliadin peptides. Four alpha-gliadin synthetic peptides that displayed a chemotactic response were elected and tested in the presence of cyclosporine H, a specific inhibitor of FPR1. Blocking of FPR1 inhibited neutrophil chemotaxis to these peptides.

Article Snippet: HEK293T cells were transfected with human FPR1 according to the manufacturer’s protocol (Origene cDNA ORF Clone, OriGene Technologies, Rockville, MD).

Techniques: Blocking Assay, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Gliadin Induces Neutrophil Migration via Engagement of the Formyl Peptide Receptor, FPR1

doi: 10.1371/journal.pone.0138338

Figure Lengend Snippet: Flow cytometry analysis of FITC-labeled gliadin peptide or fMet-Leu-Phe binding to FPR1-transfected or non-transfected HEK293T cells. (A) Kinetic binding of FITC-labeled gliadin synthetic peptide, TLPAMCNVYIPPYCTIVPFG, applied at increasing concentrations (ranging from 1 to 250 μM). Dissociation constant (K d ) was 235.1 μM, and Bmax was 922.4. (B) Kinetic binding of FITC-labeled fMet-Leu-Phe, applied at increasing concentrations (ranging from 1 to 1000 nM). Dissociation constant (K d ) was 27.6 nM, and Bmax was 261.6. (C) Competitive binding assay was performed with FITC-labeled gliadin peptide at 25.6 μM and unlabeled fMet-Leu-Phe at increasing concentrations (ranging from 10 nM to 2 mM). fMet-Leu-Phe caused a displacement of the gliadin peptide from the FPR1-transfected HEK293T cells with an IC50 of 2.04 μM. (D) Competitive binding assay was performed with FITC-labeled fMet-Leu-Phe at 100 nM and unlabeled gliadin peptide at increasing concentrations (ranging from 1 nM to 500 μM). The gliadin peptide was not capable of displacement of the fMet-Leu-Phe from the FPR1-transfected HEK293T cells. Binding to non-transfected cells and FPR1-transfected HEK293T cells is depicted with white and black circles, respectively. Each graph represents data from 3–5 independent experiments.

Article Snippet: HEK293T cells were transfected with human FPR1 according to the manufacturer’s protocol (Origene cDNA ORF Clone, OriGene Technologies, Rockville, MD).

Techniques: Flow Cytometry, Labeling, Binding Assay, Transfection, Competitive Binding Assay