Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell Sequencing Reveals Neutrophil Extracellular Traps in Association with Endotheliopathy and Immunothrombosis in Hepatitis B Virus-related Acute-on-chronic Liver Failure

doi: 10.14218/JCTH.2025.00666

Figure Lengend Snippet: (A) Visualization of differentially expressed genes and pathways in neutrophils from HBV-ACLF patients and HC using the circlize R package. (B) mRNA expression levels of TLR2 and C5aR1 in neutrophils from HC, CHB, and HBV-ACLF patients. (C) Immunofluorescence images (SYTOX Green and DAPI) in neutrophils stimulated with plasma from HC, CHB, and HBV-ACLF groups. Scale bars: 50 µm. (D) Quantification of NETs release from neutrophils stimulated with plasma from HC, CHB, and HBV-ACLF groups, measured by SYTOX Green fluorescence. (E) NETs release in the presence or absence of TLR2 or C5aR1 inhibitors. * P < 0.05, *** P < 0.001, and **** P < 0.0001. NETs, neutrophil extracellular traps; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; TLR2, toll-like receptor 2; C5aR1, complement component 5a receptor 1; CHB, chronic hepatitis B; HC, healthy controls.

Article Snippet: In selected experiments, neutrophils were preincubated with the complement component 5a receptor 1 (C5aR1) antagonist (HY-163379; MedChemExpress) or the toll-like receptor 2 (TLR2) antagonist (HY-116213; MedChemExpress) for 30 min prior to plasma stimulation.

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Fluorescence, Virus

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Experimental design of permanent left anterior descending (LAD) coronary artery ligation in Pf4^cre+^ C5ar1^fl/fl^ mice and Cre-negative littermate controls, followed by serial echocardiography and terminal analyses up to day 14. (B) Representative transverse left ventricular sections stained with TTC on day 14 after myocardial infarction, with infarcted myocardium appearing white and viable myocardium appearing red. Infarcted areas are indicated by a solid white outline (scale bar = 2 mm). (C) Quantification of infarct size expressed as percentage of left ventricle (LV). (D) Representative M-mode echocardiographic images. (E) Echocardiographic assessment of left ventricular ejection fraction (EF; left) and fractional shortening (FS; right) on day 1 and day 13 after myocardial infarction. (F) Representative immunofluorescence images of collagen I (red) and DNA (DAPI, blue) on day 14. (G) Quantification of collagen I–positive area (collagen I, % of LV section). (H) Representative CD31 (green) immunostaining with DNA staining (DAPI, blue) in the peri-infarct region on day 14. (I) Quantification of capillary density (CD31-positive area, %). Data are shown as mean ± SD; each dot represents one mouse. n = 4 mice per group for TTC and immunofluorescence analyses; n = 9–10 mice per group for echocardiography. Statistical analysis was performed using one-way ANOVA for EF and FS and two-tailed unpaired t-tests for all other comparisons. P < 0.05, P < 0.01, P < 0.001.

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Ligation, Staining, Immunofluorescence, Immunostaining, Two Tailed Test

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Representative immunofluorescence images of peri-infarct myocardium stained for Ly6G (red) and CD42b (green) with DNA staining (DAPI, blue), showing myocardial platelet–neutrophil complexes (PNCs). (B) Quantification of myocardial PNC density (mm⁻²) in the peri-infarct region (see also Supplementary Figure 10 for platelet and neutrophil infiltration). (C) Representative immunofluorescence images of peri-infarct myocardium stained for myeloperoxidase (MPO, red) and citrullinated histone H3 (H3Cit, green) with DNA (DAPI, blue), showing neutrophil extracellular traps (NETs). (D) Quantification of NET burden expressed as percentage of H3Cit⁺ area of the left ventricle (LV). (E) Flow cytometric gating strategy for platelet–neutrophil complexes (PNCs) in whole blood. Neutrophils were identified by Ly6G, and PNCs were defined as Ly6G⁺CD42b⁺ events. (F) Circulating PNCs expressed as percentage of CD42b⁺ events among Ly6G⁺ neutrophils on day 1 and day 14 after myocardial infarction in Pf4^cre+ C5ar1^fl/fl mice and Cre-negative littermate controls. (G) Spearman correlation analysis demonstrating an inverse relationship between circulating PNCs on day 1 after myocardial infarction and myocardial PNC density on day 14 across individual mice (r = −0.83, P = 0.02). Data are shown as mean ± SD; each dot represents one mouse. n = 4 mice per group for immunofluorescence analyses (A–D) and n = 9 mice per group for flow cytometric analyses (E–F). Statistical analysis was performed using two-tailed unpaired t-tests for immunofluorescence data, one-way ANOVA for flow cytometric analyses, and Spearman’s rank correlation for association analysis (G). P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Scale bars, 20 µm (A) and 10 µm (C).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Representative single-plane confocal immunofluorescence images of fixed and permeabilized resting wild-type (WT) and C5aR1-deficient platelets stained for P-selectin (CD62P, green) and CXCL4 (red), illustrating altered α-granule organization. (B) Quantification of intracellular α-granule content per platelet, expressed as total area of P-selectin–positive granules and colocalized P-selectin/CXCL4 granules, measured by confocal microscopy. Data are shown as individual platelet values pooled from n = 3 independent experiments. Outliers were identified and removed using the ROUT method (Q = 1%) prior to analysis. (C) Flow cytometric analysis of platelet surface P-selectin expression 24 h after myocardial infarction following ex vivo stimulation of whole blood with 100 nM phorbol 12-myristate 13-acetate (PMA), expressed as geometric mean fluorescence intensity (GMFI) of CD42b⁺ platelets. (D) Flow cytometric analysis of platelet integrin GPIIb/IIIa activation under the same conditions, expressed as percentage of activated GPIIb/IIIa among CD42b⁺ platelets. (E) Plasma CXCL4 concentrations after MI in Pf4^cre+^ C5aR1^fl/fl^ mice and Cre-negative littermate controls. (F) Schematic of the in vitro platelet–neutrophil co-incubation assay. WT or C5aR1-deficient platelets were stimulated with C5a and co-incubated with neutrophils, followed by confocal immunofluorescence staining for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA (DAPI) to assess NET formation, in the presence or absence of low-dose heparin or recombinant CXCL4 (rCXCL4). (G) Representative immunofluorescence images of neutrophils after co-incubation, stained for MPO (red), H3Cit (green), and DNA (DAPI, blue), illustrating NET formation under the indicated conditions (see Supplementary Figure 13 for neutrophil-intrinsic and platelet-mediated control conditions). (H) Quantification of NET formation expressed as percentage of H3Cit⁺ neutrophils. Data are shown as mean ± SD unless otherwise indicated; each dot represents one biological replicate or mouse, as indicated. For α-granule analyses (A–B), data are shown as individual platelet values pooled from n = 3 independent experiments and analyzed using two-tailed unpaired t-tests following ROUT-based outlier exclusion (Q = 1%). Flow cytometry data (C–D) were analyzed using one-way ANOVA across time points and genotypes (see also Supplementary Figures for day 14 analyses). Plasma CXCL4 measurements (E) were analyzed using two-tailed unpaired t-tests. NET formation assays (H) were analyzed using one-way ANOVA with appropriate post hoc correction. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Scale bars, 2 µm (A) and 20 µm (G).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Expressing, Ex Vivo, Fluorescence, Activation Assay, Clinical Proteomics, In Vitro, Incubation, Recombinant, Control, Two Tailed Test, Flow Cytometry

Journal: bioRxiv

Article Title: Platelet C5aR1 Aggravates Myocardial Infarction through Platelet–Neutrophil Interactions and CXCL4-Dependent NET Release

doi: 10.64898/2026.01.12.699090

Figure Lengend Snippet: (A) Experimental design for daily subcutaneous administration of the C5aR1 inhibitor PMX205 or PBS for 14 days following permanent left anterior descending (LAD) coronary artery ligation. (B) Representative TTC-stained transverse left ventricular sections on day 14 after myocardial infarction, with infarcted myocardium appearing white and viable myocardium appearing red. Infarcted areas are indicated by a solid white outline (left). Corresponding quantification of infarct size, expressed as percentage of left ventricular (LV) area (right). (C) Quantification of collagen I–positive area in immunofluorescence staining, expressed as percentage of LV area. (D) Representative M-mode echocardiographic images on day 13 after myocardial infarction. (E) Echocardiographic assessment of left ventricular ejection fraction (EF; left) and fractional shortening (FS; right) on day 1 and day 13 after myocardial infarction. (F) Representative immunofluorescence images of peri-infarct myocardium stained for Ly6G (red) and CD42b (green) with DNA (DAPI, blue), illustrating myocardial platelet–neutrophil complexes (PNCs). (G) Quantification of myocardial PNC density (mm⁻²). (H) Quantification of myocardial NET burden expressed as percentage of H3Cit⁺ neutrophils. (I) Representative immunofluorescence images of peri-infarct myocardium stained for myeloperoxidase (MPO), citrullinated histone H3 (H3Cit), and DNA (DAPI), illustrating NET deposition. (J) Representative flow cytometry plots of platelet surface P-selectin expression and activated GPIIb/IIIa (αIIbβ3) in PMA-stimulated whole blood 24 h after myocardial infarction. (K) Quantification of platelet surface P-selectin expression in PMA-stimulated whole blood 24 h after myocardial infarction. (L) Quantification of platelet GPIIb/IIIa activation in PMA-stimulated whole blood 24 h after myocardial infarction. Data are shown as mean ± SD; each dot represents one mouse. Statistical analysis was performed using two-tailed unpaired t-tests for infarct size and collagen I quantification (B, C) and for myocardial PNC and NET quantification (G, H). Echocardiographic parameters (E) and flow cytometric platelet activation analyses (K, L) were analyzed using one-way ANOVA across time points and treatment groups (see also Supplementary Information for day 14 analyses). P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Gating strategies are provided in the Supplementary Information. Scale bars, 2 mm (B) and 20 µm (F, I).

Article Snippet: For pharmacological inhibition experiments, wild-type mice only received the selective C5aR1 antagonist PMX205 (Tocris) or PBS vehicle.

Techniques: Ligation, Staining, Immunofluorescence, Flow Cytometry, Expressing, Activation Assay, Two Tailed Test

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: Upregulation of C5a/C5aR1 promotes cytokine release and drives inflammation in LN mice. (A) Schematic diagram of the animal model induction process; (B) Mouse cytokine antibody array Panel (A) showing increased C5a expression; (C) Western blot analysis of C5aR1 expression in mouse renal tissues, with β‐actin as the internal control ( n = 5); (D) Immunohistochemical analysis of C5aR1 expression in paraffin‐embedded kidney sections ( n = 5); (E) Analysis of IL‐1β and TNF‐α mRNA expression in mouse kidney tissues, with β‐actin as the internal control ( n = 5). (F) Representative H&E staining of paraffin‐embedded kidney sections ( n = 5); black arrows indicate inflammatory cell infiltration around renal tubules. Group comparisons were performed using a two‐tailed unpaired t ‐test, followed by LSD or Tukey post hoc tests to determine intergroup differences. Immunohistochemistry scores were analyzed using nonparametric tests. p > 0.05, not significant. **** p < 0.0001. H&E, hematoxylin and eosin; LN, lupus nephritis; LSD, least significant difference.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Animal Model, Ab Array, Expressing, Western Blot, Control, Immunohistochemical staining, Staining, Two Tailed Test, Immunohistochemistry

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: C5aR1A alleviates inflammation and renal dysfunction in LN mice. (A) Schematic diagram of the animal model procedure; (B) mRNA expression levels of IL‐1β and TNF‐α in mouse renal tissue, normalized to β‐actin ( n = 5); (C) PAS and H&E staining of paraffin‐embedded kidney sections ( n = 5); (D, E) Immunohistochemical analysis of IL‐1β, MCP‐1, TNF‐α, and TGF‐β expression in renal tissues at 40× magnification ( n = 5); (F) Western blot analysis of PTEN and p‐AKT in mouse kidney tissue, with GAPDH as a loading control ( n = 5); (G) Quantification of BUN and Scr levels in mouse kidney tissues ( n = 5). Statistical analysis was performed using one‐way ANOVA. p > 0.05, not significant. * p < 0.05; *** p < 0.001; **** p < 0.0001. ANOVA, analysis of variance; BUN, blood urea nitrogen; H&E, hematoxylin and eosin; LN, lupus nephritis; MCP‐1, monocyte chemoattractant protein‐1; PAS, periodic acid–Schiff; PTEN, phosphatase and tensin homolog; Scr, serum creatinine; TGF‐β, transforming growth factor‐β.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Animal Model, Expressing, Staining, Immunohistochemical staining, Western Blot, Control

Journal: Journal of Cell Communication and Signaling

Article Title: Regulation of phosphatase and tensin homolog by complement component 5a (C5a) and its receptor (C5aR1) in lupus nephritis: A novel therapeutic target

doi: 10.1002/ccs3.70055

Figure Lengend Snippet: C5a suppresses PTEN expression and enhances AKT pathway activation to promote inflammation. (A) Schematic diagram of the animal model procedure; (B) Schematic diagram of the in vitro cell experiment; (C) Protein‐protein interaction network illustrating key molecules linking C5a/C5aR11 with PTEN and the PI3K/AKT signaling pathway (confidence score = 0.15); (D) Western blot analysis of C5aR1 knockdown by three siRNAs in vitro (D1–D2) and by three shRNAs in kidney tissues in vivo (D3–D4), with GAPDH as loading control ( n = 3); (E) Western blot analysis of C5aR1 and PTEN expression after C5aR1 knockdown by siRNAs in vitro (E1–E3) and by shRNAs in kidney tissues in vivo (E4–E6), with GAPDH as loading control ( n = 3). (F–H) Results of the human/mouse AKT pathway phosphorylation antibody array C1 (RayBiotech) showing the expression of BAD, PRAS40, and PTEN. Group comparisons were performed using a two‐tailed unpaired t ‐test or one‐way ANOVA. p > 0.05, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ANOVA, analysis of variance; BAD, Bcl‐2‐associated death promoter; PTEN, phosphatase and tensin homolog.

Article Snippet: The LN + C5aRA1 group received PMX53, a C5aR1 antagonist (10 mg/kg; HY‐106178, MedChemExpress), once daily by oral gavage.

Techniques: Expressing, Activation Assay, Animal Model, In Vitro, Western Blot, Knockdown, In Vivo, Control, Phospho-proteomics, Ab Array, Two Tailed Test

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: A Representative images of C5aR1 staining in TMAs for prostate, endometrial, and colorectal normal and tumour tissues. A zoomed in region of the TMA is shown to the right. Scale bar, 100 µm. B H-score of staining for TMAs in ( A ) is shown. Individual dots show data for different cores analysed for each patient. 3 prostate, 4 endometrial and 5 colorectal cancer patient samples were analysed. C Pearson’s correlation of C5aR1 mRNA expression and Ahmed Hypoxia signature in TCGA colorectal cancer and glioblastoma samples. R score and p -value are shown. D , E HCT116 ( D ) and LN229 ( E ) cells were cultured under the indicated oxygen conditions for 24 h and 16 h, respectively, and subjected to qRT-PCR. n = 3. Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by one-way ANOVA with Dunnett’s test. F , G Serial sections of HCT116 spheroids treated with EF5 were stained with the indicated antibodies; C5aR1 (red), a hypoxia marker, EF5 (green), or DAPI (blue). The dotted line represents the estimated outside edge of the EF5-positive regions. Scale bar, 50 µm.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Staining, Expressing, Cell Culture, Quantitative RT-PCR, Marker

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by two-tailed paired Student’s t -test ( A ), two-tailed unpaired Student’s t -test ( B − D ), two-way ANOVA with uncorrected Fisher’s LSD test ( E – G and I ), or one-way ANOVA with Dunnett’s test ( H ). A HIF-1α- KO and WT HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to qRT-PCR. n = 3 and 4. B – D HCT116 cells were treated with either 200 µM cobalt chloride (CoCl 2 ) for 24 h ( B ), 10 µg/mL tunicamycin (Tuni) for 24 h ( C ), 2 µM thapsigargin (Thap) for 16 h ( D ), or its vehicle (Ctrl), and subjected to qRT-PCR. n = 3. E – G HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) in the presence of either IRE1-inhibitor (E), PERK-inhibitor ( F ), or ATF6-inhibitor ( G ), and subjected to qRT-PCR. n = 3. H HCT116 cells were transfected with the indicated siRNA or scramble siRNA (siScr), cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to qRT-PCR. n = 4. I After simultaneously silencing XBP1, ATF4, and ATF6 using siRNA mixtures, HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to qRT-PCR. n = 3. J Enrichment Analysis with public ChIP-seq data on ChIP-Atlas website is shown ( https://chip-atlas.org/peak_browser ). Colours represent copy numbers of the indicated transcription factors binding to the C5aR1 gene locus in all cell lines. K Pearson’s correlation of C5aR1 mRNA expression and Xhu UPR signature in TCGA colorectal cancer and glioblastoma samples. R score and p -value are shown. L , M Serial sections of HCT116 spheroids treated with EF5 ( L ) or HCT116 tumour xenografts ( M ) were stained with the indicated antibodies; ( L ) Section 1, C5aR1(red), BiP (green), or DAPI (blue); Section 2, BiP (red), EF5 (green), or DAPI (blue). (M) C5aR1 (red), BiP (green), or DAPI (blue). Scale bar, 50 µm.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Quantitative RT-PCR, Transfection, ChIP-sequencing, Binding Assay, Expressing, Staining

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by two-way ANOVA with uncorrected Fisher’s LSD test ( C , D , and F ) or two-tailed paired Student’s t -test ( G – J ). A , B HCT116 cells were transfected with either siC5aR1 or siScr ( A ) or with either pcDNA3.1/C5aR1-GFP (C5aR1) or its empty vector (EV) ( B ), cultured under normoxia or hypoxia (<0.1% O 2 ) for the indicated periods, and subjected to immunoblotting. C RKO cells were transfected with siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to immunocytochemistry and apoptosis assay. C5aR1 (red), Phalloidin (green), or DAPI (blue). Scale bar, 10 µm. n = 3. D HCT116 cells were transfected with either siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to apoptosis assay. n = 3. E HCT116 cells were transfected with either siRNA against C5 (siC5) or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for the indicated periods, and subjected to immunoblotting. F RKO cells were transfected with either siC5 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to apoptosis assay. n = 3. G , H HCT116 cells were transfected with either siC5aR1 or siScr ( G ) or with either pcDNA3.1/C5aR1-GFP (C5aR1) or EV ( H ), cultured under normoxia or hypoxia (<0.1% O 2 ) for 40 h ( G ) and 32 h ( H ), respectively, and subjected to cell viability assay. n = 3. I , J HCT116 ( I ) and RKO ( J ) cells were transfected with either siC5aR1 or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h and subjected to clonogenic survival assay. n = 3.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Immunocytochemistry, Apoptosis Assay, Viability Assay, Clonogenic Cell Survival Assay

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by one-way ANOVA with Dunnett’s test ( A , B , and E ; DMSO vs JPE-1375 and Avacopan), two-way ANOVA with uncorrected Fisher’s LSD test ( C , H ), and two-tailed paired Student’s t -test ( E ; Water vs PMX205), two-way ANOVA with Tukey test ( F ). A , B HCT116 cells were pretreated for 8 h with the indicated dose of C5aR1 antagonists: PMX205, JPE-1375 or Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 40 h and subjected to cell viability assays. n = 3. C – E HCT116 cells were pretreated for 8 h with 12 μM PMX205, 10 μM JPE-1375 or 2 μM Avacopan. Cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h ( C and E ) and 16 h ( D ), and were subjected to apoptosis assay ( C ), immunoblotting ( D ), and clonogenic survival assay ( E ), respectively. n = 3. F Diameters of HCT116 spheroids were measured daily after the administration of the indicated drug. Representative bright-field microscopy images (left) and changes in diameter (right) of spheroids are shown. n = 12 ( G , H ) HCT116 spheroids were treated with vehicle or Avacopan and EF5. Sections were subjected to TUNEL assay and immunohistochemistry. Apoptosis (red), EF5 (green), or DAPI (blue). Scale bar, 50 µm. Representative images ( G ) and red fluorescence score in EF5 positive and negative regions ( H ) are shown. n = 6–7.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Apoptosis Assay, Western Blot, Clonogenic Cell Survival Assay, Microscopy, TUNEL Assay, Immunohistochemistry, Fluorescence

Journal: Cell Death & Disease

Article Title: UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment

doi: 10.1038/s41419-025-07862-z

Figure Lengend Snippet: For the whole figure: Individual biological replicates (large points) represent the average of the technical replicates (small points). p -values were calculated using biological replicates by two-way ANOVA with uncorrected Fisher’s LSD test ( E and F ), and two-tailed paired Student’s t -test ( B and D ). A Schematic representation of experimental design for B and D–F. Created in BioRender.com. B RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 16 h, and subjected to FACS, with (right) or without (left) permeabilisation. n = 3. C RKO cells were cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to immunocytochemistry. C5aR1 (red), Phalloidin (green), or DAPI (blue). Scale bar, 10 µm. D After treatment with 10 µg/mL tunicamycin (Tuni) or vehicle (DMSO) for 24 h, HCT116 cells were subjected to FACS with (right) or without (left) permeabilisation. n = 3. E HCT116 cells were cultured under normoxia or hypoxia (<0.1% O 2 ) in the presence of 100 µM Dynasore and subjected to FACS with (right) or without (left) permeabilisation. n = 3. F HCT116 cells were transfected with either siRNA against C5 (siC5) or siScr, cultured under normoxia or hypoxia (<0.1% O 2 ) for 24 h, and subjected to FACS with (right) or without (left) permeabilisation. n = 3. G Working model: In hypoxic cancer cells, UPR-induced C5aR1 is internalised and accumulates by endocytosis. Intracellular C5aR1 contributes to cancer cell survival by modulating autophagy and apoptosis under hypoxia. To effectively target the C5a-C5aR1 axis in the TME, cell permeable C5aR1 inhibitors may be more effective.

Article Snippet: Before hypoxic culture, cells were pretreated with IRE1α inhibitor (4μ8c, Sigma-Aldrich, SML0949), PERK inhibitor (AMG PERK 44, Tocris, 5517), ATF6 inhibitor (Ceapin-A7, Sigma-Aldrich, SML2330), and Dynasore (Sigma-Aldrich, D7693) for 1 h, or with C5aR1 antagonists/inhibitors, PMX205 (Tocris, 5196), JPE-1375 (MedChem Express, HY-148141) and Avacopan (Cayman Chemical, CAY36639 ) for 8 h, respectively.

Techniques: Two Tailed Test, Cell Culture, Immunocytochemistry, Transfection

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: C5aR1 is mainly localized in unmyelinated fibers. Longitudinal and cross‐sections of human sural nerves were labeled for C5aR1 (red, A; green, D), Caspr (green, B), and NCAM (E, red). Double‐stained images are merged (C, F). Teased mouse sciatic nerve images of labeled C5aR1 (red, G, J) and Caspr (green, H). Double‐stained mouse sciatic fibers were merged (I). Peripherin (green, K) merged (L). Caspr‐contactin‐associated protein; NCAM‐neural cell adhesion molecule; n = 10 mice; Scale bar 10, 20 μM.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Labeling, Staining

Journal: Journal of Neurochemistry

Article Title: Complement C3a and C5a Receptors Are Presented in Mouse Sciatic and Human Sural Nerves and Selectively Modulate the Neuronal Function of Large‐Caliber Fibers in Mice

doi: 10.1111/jnc.70129

Figure Lengend Snippet: mRNA and protein expression in the mouse sciatic nerve. Quantitative PCR (qPCR) analysis and western blotting were performed on sciatic nerves from 13‐week‐old mice ( n = 6 nerves). Intrinsic expression of the complement receptors and factors is seen (A). The outcomes are presented relative to hypoxanthine guanine phosphoribosyltransferase expression using the 2 −ΔCT method calculation method. Proteins C3aR and C5aR1 are present in mouse sciatic nerves ( n = 6 nerves) (B, C). HPRT‐hypoxanthine guanine phosphoribosyltransferase; ** p < 0.01.

Article Snippet: The C5aR1 agonist BM213 (1 μM, Cat. No. HY‐145237, MedChemExpress), the C5aR1 antagonist PMX205 (1 μM, Cat. No. 13607, Cayman Chemicals, Ann Arbor, Michigan, USA) and the combination were applied at the same final concentrations as described above.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot