Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Selected potential paclitaxel targets and their predicted affinity.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques:

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: ( A ) Binding of paclitaxel on immobilized C5aR1, C5aR2, C5a, and C5a des-Arg. ( B ) Dose-dependent binding of paclitaxel on immobilized C5aR1. ( C , D ) Co-injection of paclitaxel and C5aR1 on immobilized C5a and relative IC50. ( E , F ) Co-injection of paclitaxel and C5aR1 on immobilized C5a des-Arg and relative IC50. G , H Co-injection of paclitaxel and C5aR1 on immobilized monoclonal antibody anti-C5aR1 and relative IC50.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques: Binding Assay, Injection

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Representative trace of electrophysiological recordings in DRG cells maintained under basal conditions ( A ), and in cells challenged with paclitaxel (10 nM) (PAC) ( B ), paclitaxel+DF3966A (1 µM) ( C ), C5a (100 nM) ( D ) or C5a + DF3966A ( E ). Quantitative evaluation of action potential firing rate in DRG exposed to paclitaxel alone or in combination with DF3966A ( F ), and C5a alone or in combination with DF3966A ( G ). DRG-derived neurons were cultured for 7 days. Cells were treated for 1 min 30 s (short) or 5 min (long). ** P < 0.005 and * P < 0.05 vs respective basal condition; + P < 0.05 vs paclitaxel or C5a. Data are mean ± SEM of n = 3 different experiments.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques: Derivative Assay, Cell Culture

Journal: Cell Death & Disease

Article Title: Paclitaxel binds and activates C5aR1: A new potential therapeutic target for the prevention of chemotherapy-induced peripheral neuropathy and hypersensitivity reactions

doi: 10.1038/s41419-022-04964-w

Figure Lengend Snippet: Proteome profiles of cytokines released from RAW 264.7 ( A , B ) and human ( C , D ) macrophages challenged with C5a ( A , C ) or paclitaxel ( B , D ) alone or in combination with a C5AR1 antibody (anti C5AR), Avacopan or DF3966A. RAW 264.7 and human macrophages cells were treated with C5a (10 nM), or paclitaxel (PAC) (10 nM), or DF3966A (1 μM) or Avacopan (1 μM) and C5aR1 antibody (1 μM) for 24 h. Data are mean ± SEM of n = 3 different experiments.

Article Snippet: The reagents that have been used are: recombinant human C5a anaphylotoxin chemotactic receptor 2 (C5AR2) (CUSABIO TECHNOLOGY, Houston, TX, US), recombinant protein of human complement component 5a receptor 1 (C5AR1) (Origene, Rockville, MD, US), anti-6X His tag antibody (Abcam, Cambridge, UK), Anti-Myc/c-Myc Antibody (9E10) (Santa Cruz Biotechnology, Dallas, Texas, US) and CD88 (clone S5/1) antibody (344302, Biolegend, San Diego, California, US), complement C5a des-Arg, Human (Merck, Burlington, Massachusetts, USA).

Techniques:

Journal: Cell host & microbe

Article Title: Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

doi: 10.1016/j.chom.2017.11.009

Figure Lengend Snippet: Figure 2. Rapid PMN Intravascular Chemotaxis to Sequestered C. albicans Is Complement Dependent (A) Lung C5a levels were determined using ELISA in control and C3/ animals, following i.v. C. albicans. Two-way ANOVA, Sidak’s multiple comparison. (B) Using intravital microscopy, PMN chemotactic behaviors in C3/ and anti-C5aR mAb-treated mice were observed. Images shown are 10 min after C. albicans injection. Red arrows highlight yeast not recognized by PMN. Scale bar represents 30 mm. (C) Quantification of PMN chemotaxing to pathogens in C3/ and C5a receptor-blocked mice. One-way ANOVA, Dunnett’s multiple comparison. (D) The percentage of C. albicans phagocytosed during the initial 10 min of intravenous administration in control, C3/, and anti-C5aR mAb-treated mice using pulmonary intravital microscopy. t tests were used to compare C57BL/6 and anti-C5aR mAb treated (*) or C3/ (#) mouse time points. (E and F) C. albicans CFU was quantified in organs at (E) 1 hr and (F) 24 hr after injection with 1 3 106 C. albicans in mice treated with anti-C5a receptor antibodies or isotype. (G) Clinical sepsis scores of isotype and anti-C5aR mAb antibody treated mice 24 hr after injection with 1 3 106 C. albicans. A score of 21 is the clinical endpoint for euthanization. For (A)–(F), at least three individual experiments were performed; for (G) three individual experiments were performed. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-Ly6G (clone 1A8) BioLegend RRID: AB_2563207 (BioLegend Cat. No. 127636) Anti-CD31 (clone MEC13.3) BioLegend RRID: AB_2161030 (BioLegend Cat. No. 102515) Anti-CD45 (clone 30-F11) BioLegend RRID: AB_493532 (BioLegend Cat. No. 103121) Anti-CD49b (clone HMa2) BD Bioscience Catalog No. 558295 Anti-C5aR (CD88, clone 20/70) BioLegend RRID: AB_2067286 (BioLegend Cat. No. 135804) Purified anti-mouse Ly6G (clone 1A8) BioXCell BP0075-1 Anti-TER119 Biolegend RRID: AB_528961 (BioLegend Cat. No. 116218) Anti-human CD15 (clone W6D3) BioLegend RRID: AB_756018 (BioLegend Cat. No. 323012) Anti-human CD45 (clone 2D1) R&D Systems Catalog # FAB1430G Anti-human C5aR (CD88, clone S5/1) BioLegend RRID: AB_2259318 (BioLegend Cat. No. 344302) Bacterial and Virus Strains C. albicans (strain ATCC58716) ATCC LUMC-101 Chemicals, Peptides, and Recombinant Proteins Zymosan A (S. cerevisiae) BioParticles ThermoFisher Z23373 Escherichia coli (K-12 strain) BioParticles ThermoFisher E13231 SYTO 9 Green Fluorescent Nucleic Acid Stain ThermoFisher S34854 Compstatin Tocris Cat. No. 2585 LY293111 Cayman Chemical Company CAS N 161172-51-6 Recombinant mouse C5a R&D Systems 2150-C5-025 Critical Commercial Assays LTB4 Parameter Assay Kit R&D Systems KGE006B EasySep Mouse Biotin Positive Selection Kit STEMCELL Technologies Catalog # 18556 Mouse Complement C5a PicoKine ELISA Kit Boster Biological Technology EK0987 Polydimethylsiloxane (PDMS) microfluidic devices SynvivoBio Cat # 104001 Experimental Models: Cell Lines human lung microvascular endothelial cells HLMEC Lonza CC-2527 Experimental Models: Organisms/Strains C57BL/6J The Jackson Laboratory Stock No: 000664 LTB4R knockout (B6.129S4-Ltb4r1tm1Adl/J) The Jackson Laboratory Stock No: 008102 C3 / ; (B6;129S4-C3tm1Crr/J) Gift from Paul Kubes N/A CD11b / ; (B6.129S4-Itgamtm1Myd/J) Gift from Paul Kubes N/A CD11a / ; (B6.129S7-Itgaltm1Bll/J) Gift from Paul Kubes N/A LysM-GFP Gift from Paul Kubes N/A Ly6G-cre (C57BL/6-Ly6g(tm2621(CretdTomato)Arte)) Gift from Matthias Gunzer N/A Software and Algorithms Volocity PerkinElmer N/A Leica LAS Leica N/A Prism 7 software GraphPad V7.02 FlowJo FlowJo V10.1 e1 Cell Host & Microbe 23, 1–13.e1–e4, January 10, 2018

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Comparison, Intravital Microscopy, Injection

Journal: Frontiers in Immunology

Article Title: P-Rex1 controls phagocytosis and the killing of bacteria by murine neutrophils independently of its catalytic activity

doi: 10.3389/fimmu.2025.1591006

Figure Lengend Snippet: P-Rex1 mediates the killing of S. aureus by neutrophils independently of its catalytic Rac-GEF activity, whereas chemotaxis, ROS, and NETs require its Rac-GEF activity. (A) Bactericidal activity. Purified neutrophils from Prex1 –/– (red squares), Prex1 GD (green triangles), and wild type mice (grey circles) were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before incubation with serum-opsonized S. aureus for 90 min at a ratio of 1.5 bacteria per neutrophil. Heat-killed neutrophils were used as negative controls. Surviving bacteria were grown overnight and CFU enumerated. The % killing of bacteria by live neutrophils compared to heat-killed controls is plotted. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are one-way ANOVA with Tukey’s multiple comparisons test on log-transformed raw data; black p-values are significant, grey p-values non-significant. (B) Chemotaxis. Bone marrow cells from Prex1 –/– , Prex1 GD , and wild type mice were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min before being stimulated with 3 nM C5a in transwell filters for 40 min, or mock stimulated. Transmigrated cells were analyzed by flow cytometry in parallel to control cells, using Ly6G hi /Mac1 hi staining to identify neutrophils. Data are mean ± SEM of 3–4 independent experiments; each symbol represents the mean of one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons test on raw data. (C) fMLP-stimulated ROS production. Purified neutrophils as in (A) were primed with 1 μg/ml LPS for 90 min and then stimulated with 3 µM fMLP (filled symbols), or mock-stimulated (open symbols). ROS production was measured by real-time chemiluminescence assay with luminol and HRP for extra- and intracellular ROS. Left-hand panel shows luminometer traces from one representative experiment; right-hand panel shows the quantification as AUC over 2 min. Data are mean ± SEM of 3–5 independent experiments; each symbol represents the mean AUC from one experiment. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on log-transformed raw data. (D) S. aureus -stimulated intracellular ROS. Neutrophils were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min in the presence of 50 units/ml SOD and 2000 units/ml catalase to scavenge extracellular ROS and were then stimulated with S. aureus at a ratio of 10 bacteria per neutrophil (filled symbols), or mock-stimulated (open symbols). ROS production was measured as in (C) except without HRP and in the presence of SOD and catalase, and quantification was done over 60 min. Data are mean ± SEM of 4 independent experiments; statistics are two-way ANOVA with Sidak’s multiple comparisons tests. (E) Formation of NETs. Neutrophils were seeded onto glass slides and allowed to adhere for 30 min before stimulation with serum-opsonised S. aureus at a ratio of 10 bacteria per neutrophil (closed symbols), or mock stimulation (open symbols). Non-cell permeable Sytox Green and cell-permeable Hoechst 33342 DNA dyes were added to samples 15 min before the end of the incubation, and cells were live-imaged by wide-field microscopy. Left-hand panel shows representative images from one experiment after 120 min stimulation or mock stimulation. Insets are magnifications of the indicated areas. Red arrows highlight NETs, white arrows dead cells without NETs. Right-hand panel shows quantification of NETs by ImageJ. Data are mean ± SEM of 3–4 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons tests on raw data; significant p-values between and Prex1 –/– and wild type are indicated in red, and between Prex1 GD and wild type in green. For all panels, closed symbols show stimulated cells, open symbols mock-treated cells.

Article Snippet: Cells were primed with 20 ng/ml TNFα and 50 ng/ml GM-CSF for 45 min at 37°C, pipetted into transwell filters (400 μl/filter) in a 24-well plate containing HBSS ++++ (600 μl/well) in the presence or absence of 3 nM complement factor C5a anaphylatoxin (C5a, R&D Systems, 2037-C5-025), and incubated for 40 min at 37°C.

Techniques: Activity Assay, Chemotaxis Assay, Purification, Incubation, Bacteria, Transformation Assay, Flow Cytometry, Control, Staining, Chemiluminescence Immunoassay, Microscopy

Journal: Biology

Article Title: Plasma Proteomic Changes of Atherosclerosis after Exercise in ApoE Knockout Mice

doi: 10.3390/biology11020253

Figure Lengend Snippet: The differentially proteins between WD and WD EX groups.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary rabbit monoclonal antibody against complement C5 (PA2308, Boster, CA, USA, 1:1000) and then horseradish peroxidase–conjugated secondary antibody was used.

Techniques: Protease Inhibitor, Coagulation

Journal: Biology

Article Title: Plasma Proteomic Changes of Atherosclerosis after Exercise in ApoE Knockout Mice

doi: 10.3390/biology11020253

Figure Lengend Snippet: Effects of exercise on ( a ) aortic complement factor C5 expression and ( b ) quantitative analysis of the complement C5 IOD/area in ApoE knockout mice. Quantification of complement C5 staining and representative images. DAB-specific threshold selection (in red) from selected aortic root areas was performed using ImageJ, and total selective area was quantified and statistically analyzed. One-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis, and *** p < 0.001 represents the significance between ND and WD groups. ## p < 0.01 represents the significance between WD and WD EX groups. n.s. = No significant difference.

Article Snippet: Then, the sections were incubated overnight at 4 °C with primary rabbit monoclonal antibody against complement C5 (PA2308, Boster, CA, USA, 1:1000) and then horseradish peroxidase–conjugated secondary antibody was used.

Techniques: Expressing, Knock-Out, Staining, Selection