Journal: Aging Cell

Article Title: Ghrelin Alleviates Aging‐Related Cognitive Impairment by Regulating Autophagy‐Related Signaling via the SHBG / JNK1 /Beclin1 Axis in Microglia

doi: 10.1111/acel.70732

Figure Lengend Snippet: The information on the antibody.

Article Snippet: C5AR1 , 1:2000 , 21316‐1‐AP , Rabbit , Proteintech , USA.

Techniques:

Journal: Aging Cell

Article Title: Ghrelin Alleviates Aging‐Related Cognitive Impairment by Regulating Autophagy‐Related Signaling via the SHBG / JNK1 /Beclin1 Axis in Microglia

doi: 10.1111/acel.70732

Figure Lengend Snippet: Network pharmacology and molecular docking suggest SHBG as a candidate mediator of ghrelin action. (A) Venn diagram showing the overlap between predicted ghrelin‐ and CI‐related targets. (B) PPI network of shared drug‐disease targets. (C) MCODE clustering analysis of the drug‐disease targets, identifying gene clusters and core genes. (D) Western blot detection of SHBG, SRC, C5AR1, KLF4, and BRCA1 expression in hippocampal tissue from mice in the control, D‐gal, and D‐gal + Ghrelin groups. (E) Subcellular localization of SHBG in BV2 cells identified by immunofluorescence (magnification: ×400, scale bar = 25 μm). (F) Molecular docking analysis of ghrelin with human SHBG. (G) Co‐IP assay validating the interaction between ghrelin and SHBG in primary microglia. (H) Western blot analysis of SHBG expression in hippocampal tissue from mice in the Control, D‐gal, D‐gal + Ghrelin‐L, and D‐gal + Ghrelin‐H groups. ns, p > 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: C5AR1 , 1:2000 , 21316‐1‐AP , Rabbit , Proteintech , USA.

Techniques: Docking Assay, Analysis, Western Blot, Expressing, Tissue, Control, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: A novel long-acting C5a-blocking cyclic peptide prevents sepsis-induced organ dysfunction via effective blockade of the inflammatory cascade

doi: 10.1038/s41392-025-02457-8

Figure Lengend Snippet: Validation of the binding affinity and blocking specificity of C5a-blocking Cp1. a SPR analysis of the mouse C5a protein with linear peptides K1, K2, K3 or K4. b Flow cytometry analysis of competitive inhibition assays for C5a-blocking linear peptides K1, K2, K3, and K4 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. c The inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on mouse neutrophil migration determined by chemotaxis assay at a dose of 10 ng per well, n = 5. d The in vivo inhibitory efficiency of C5a-blocking linear peptides K1, K2, K3 and K4 on plasma proinflammatory cytokines and chemokines determined by ELISA at a dose of 100 μg/20 g via i.v. injection, n = 3. e SPR analysis of cyclic-peptide Cp1 with mouse C5a, human C5a, mouse C5a-desArg or mouse C5. f Flow cytometry analysis of competitive inhibition assays for Cp1 and Cp3 at high (1 μg/mL) or low (10 ng/mL) concentrations, n = 3. g Cyclic peptide Cp1, Cp3 or anti-C5 antibody (eculizumab)-induced erythrocyte hemolysis rate quantified by hemoglobin absorbance at 540 nm, n = 3. h Validation of Cp1-specific blockade of C5a-C5aR1 detected by flow cytometry. PMNs (without TNF-α stimulation) were preincubated with C5aR1-antibody, C5aR2-antibody, Cp1, Cp1 + C5aR1-antibody or Cp1 + C5aR2-antibody at room temperature for 0.5 h, followed by incubation with Cy5-C5a at 4 °C for 0.5 h in the dark, n = 3. The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: PMNs isolated by flow cytometric sorting from Oribiotech Co., Ltd. (Shanghai, China) were preincubated with C5aR1-antibody (5 μg), C5aR2-antibody (5 μg), Cp1 (10 μg), Cp1 (10 μg)+C5aR1-antibody (5 μg) or Cp1 (10 μg)+C5aR2-antibody (5 μg) at room temperature for 0.5 h, followed by incubation with 10 ng of Cy5-labeled human C5a (MCE, Shanghai, China) at 4 °C for 0.5 h in the dark.

Techniques: Biomarker Discovery, Binding Assay, Blocking Assay, Flow Cytometry, Inhibition, Migration, Chemotaxis Assay, In Vivo, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Injection, Incubation

Journal: British Journal of Cancer

Article Title: Inhibition of C5a-C5aR1 axis suppresses tumour progression by enhancing antitumour immunity and chemotherapeutic effect in pancreatic ductal adenocarcinoma

doi: 10.1038/s41416-025-03185-0

Figure Lengend Snippet: a High C5a expression in human PDAC cell cytoplasm ( n = 90). Original magnification: ×200. b Low C5a expression in human PDAC cell cytoplasm ( n = 40). Original magnification: ×200. c High C5aR1 expression in human PDAC cell cytoplasm ( n = 62). Original magnification: ×200. d Low C5aR1 expression in human PDAC cell cytoplasm ( n = 68). Original magnification: ×200. e High C5aR2 expression in human PDAC cell cytoplasm ( n = 102). Original magnification: ×200. f Low C5aR2 expression in human PDAC cell cytoplasm ( n = 28). Original magnification: ×200. g – i Images of the immunofluorescence (IF) staining of DAPI (blue), C5a (green), and C5aR1 (red) expression in the human PDAC cytoplasm. C5a and C5aR1 are expressed in the PDAC cell cytoplasm, and margin sites were observed (arrowheads). Original magnification: ×200. j – l Images of IF staining of DAPI (blue), C5a (green), and C5aR2 (red) expression in the human PDAC cytoplasm. C5a and C5aR2 are expressed in the PDAC cell cytoplasm, and margin sites were observed (arrowheads). Original magnification: ×200. m Kaplan–Meier analyses for OS of PDAC patients based on PDAC cell cytoplasmic C5a-C5aR1 axis (C5a-C5aR1 c-axis). The high C5a-C5aR1 c-axis group presented significantly shorter OS ( p = 0.0078, log-rank test) than low C5a-C5aR1 c-axis group. n Kaplan–Meier analyses for OS of PDAC patients based on PDAC cell cytoplasmic C5a-C5aR2 axis (defined: C5a-C5aR2 c-axis). The Low C5a-C5aR2 c-axis group presented significantly shorter OS ( p = 0.049, log-rank test) than the High C5a-C5aR2 c-axis group. o Kaplan–Meier analyses for OS of PDAC patients based on PDAC cell cytoplasmic C5a-C5aR1 c-axis and C5a-C5aR2 c-axis combination. The high C5a-C5aR1 c-axis and low C5aR2 c-axis group presented significantly shorter OS than other groups ( p = 0.0001, log-rank test).

Article Snippet: The detailed information of C5a, C5/C5b, C5aR1, and C5aR2 antibodies was indicated as follows; Human C5a mouse monoclonal antibody (D2): cat. no. MA5-43875, Company: Thermo Fisher Scientific Japan, Degree of dilution for IHC and IF: 1:100; Human C5/C5b monoclonal antibody: cat. no. HA255621 , Company: ABclonal, Degree of dilution for IHC: 1:100; Human C5aR1 rabbit polyclonal antibody: cat. no. A1900, Company: ABclonal Hubei China, Cross-reactivity: mouse, rat, Degree of dilution for IHC and IF: 1:150; Human C5aR2 rabbit polyclonal antibody: cat. no. HPA016629, Company: Merck Ltd./Sigma-Aldrich Japan G.K., Degree of dilution for IHC and IF: 1:75.

Techniques: Expressing, Immunofluorescence, Staining

Journal: British Journal of Cancer

Article Title: Inhibition of C5a-C5aR1 axis suppresses tumour progression by enhancing antitumour immunity and chemotherapeutic effect in pancreatic ductal adenocarcinoma

doi: 10.1038/s41416-025-03185-0

Figure Lengend Snippet: a Western blot analysis of C5a and C5aR1, C5aR2 expression in various human pancreatic cell lines, including cell lines of primary PDAC (BxPC-3, MIA PaCa-2, PANC-1, Capan-2), metastatic PDAC ascites (AsPC-1), lymph node metastasis (Hs766T), and PDAC liver metastases (CFPAC-1, Capan-1). b Western blot analysis of the C5aR1 knockdown with C5aR1siRNA transfection in MIA PaCa-2, BxPC-3 cells. The expression of C5aR1 in C5aR1siRNA1, 2-transfected cells was decreased by normalisation with β-actin compared to the control siRNA cells. c Proliferation assays using C5aR1siRNAs-transfected cells. The absorbance of MIA PaCa-2 and BxPC-3 cells, which had undergone C5aR1 knockdown with C5aR1siRNA1, 2 transfection, showed significant reduction after 48 h in both cells when compared to the control siRNA group (* p < 0.05, ** p < 0.01, Mann–Whitney–Wilcoxon test). d The efficacy of C5aR1 selective inhibitor (CCX168) in proliferation assays. The absorbance of MIA PaCa-2 and BxPC-3 cells, which had been seeded with CCX168, showed significant reduction after 48 h in both cells when compared to the control group (* p < 0.05, ** p < 0.01, Mann–Whitney–Wilcoxon test). e Western blot analysis with C5aR1siRNA-transfected cells (MIA PaCa-2, BxPC-3) showed decreasing expression of p-PI3K, p-AKT, and p-mTOR by normalisation with β-actin compared to the control cells, whereas the expression of t-PI3K, t-AKT, and t-mTOR showed no difference compared to the control cells. f The analyses of proliferation using Gemcitabine (Gem) and CCX168. The absorbance of MIA PaCa-2 and BxPC-3 cells, which had been seeded with CCX168, showed significant reduction after 48 h in both cells when compared to the control group (* p < 0.05, ** p < 0.001, Mann–Whitney–Wilcoxon test). In addition, proliferation in MIA PaCa-2 cells was significantly inhibited after 48 h in the Gem and CCX168 combined group compared to the Gem or CCX168 group. g , h Apoptosis assay utilising annexin V and PI using MIA PaCa-2 cells. The number of early apoptotic cells that were positive for Annexin V, but negative for PI, was significantly increased in the group treated with Gem in combination with C5aR1siRNA transfection or CCX168 group compared to Gem group (* p < 0.05, ** p < 0.001, Mann–Whitney–Wilcoxon test).

Article Snippet: The detailed information of C5a, C5/C5b, C5aR1, and C5aR2 antibodies was indicated as follows; Human C5a mouse monoclonal antibody (D2): cat. no. MA5-43875, Company: Thermo Fisher Scientific Japan, Degree of dilution for IHC and IF: 1:100; Human C5/C5b monoclonal antibody: cat. no. HA255621 , Company: ABclonal, Degree of dilution for IHC: 1:100; Human C5aR1 rabbit polyclonal antibody: cat. no. A1900, Company: ABclonal Hubei China, Cross-reactivity: mouse, rat, Degree of dilution for IHC and IF: 1:150; Human C5aR2 rabbit polyclonal antibody: cat. no. HPA016629, Company: Merck Ltd./Sigma-Aldrich Japan G.K., Degree of dilution for IHC and IF: 1:75.

Techniques: Western Blot, Expressing, Knockdown, Transfection, Control, MANN-WHITNEY, Apoptosis Assay

Journal: British Journal of Cancer

Article Title: Inhibition of C5a-C5aR1 axis suppresses tumour progression by enhancing antitumour immunity and chemotherapeutic effect in pancreatic ductal adenocarcinoma

doi: 10.1038/s41416-025-03185-0

Figure Lengend Snippet: a High C5a expression in human PDAC stroma ( n = 92). Original magnification: ×200. b Low C5a expression in human PDAC stroma ( n = 38). Original magnification: ×200. c High C5aR1 expression in human PDAC stroma ( n = 65). Original magnification: ×200. d Low C5aR1 expression in human PDAC stroma ( n = 65). Original magnification: ×200. e In the Low C5a-C5aR1 s-axis group, the proportion of the Low C5a-C5aR1 c-axis was significantly higher than in the High C5a-C5aR1 s-axis group ( p = 0.0076; Welch’s t test). f Kaplan–Meier analyses of the OS of patients with PDAC based on the C5a-C5aR1 stromal axis (C5a-C5aR1 s-axis). The C5a-C5aR1 s-axis group had a significantly shorter OS than the Low C5a-C5aR1 s-axis group ( p = 0.0007, log-rank test). g – i Images of IF staining of DAPI (blue), C5a (green), and C5aR1 (red) expression in human PDAC stroma. C5a and C5aR1 were expressed in the PDAC stroma and margin sites were observed (arrowheads). Original magnification: ×200. j , k IHC for CD8 presenting CD8 + cytotoxic T cells in the PDAC stoma. Original magnification: ×40, ×400. l , m IHC for CD11b presenting MDSCs in PDAC stomas. Original magnification: ×40, ×400. n The number of CD8 + T cells was significantly lower in the C5a-C5aR1 s-axis than in the Low C5a-C5aR1 s-axis (** p < 0.01, Mann–Whitney–Wilcoxon test). o The number of CD11b + MDSCs was significantly higher in the C5a-C5aR1 s-axis than in the Low C5a-C5aR1 s-axis (** p < 0.01, Mann–Whitney–Wilcoxon test).

Article Snippet: The detailed information of C5a, C5/C5b, C5aR1, and C5aR2 antibodies was indicated as follows; Human C5a mouse monoclonal antibody (D2): cat. no. MA5-43875, Company: Thermo Fisher Scientific Japan, Degree of dilution for IHC and IF: 1:100; Human C5/C5b monoclonal antibody: cat. no. HA255621 , Company: ABclonal, Degree of dilution for IHC: 1:100; Human C5aR1 rabbit polyclonal antibody: cat. no. A1900, Company: ABclonal Hubei China, Cross-reactivity: mouse, rat, Degree of dilution for IHC and IF: 1:150; Human C5aR2 rabbit polyclonal antibody: cat. no. HPA016629, Company: Merck Ltd./Sigma-Aldrich Japan G.K., Degree of dilution for IHC and IF: 1:75.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Inhibition of C5a-C5aR1 axis suppresses tumour progression by enhancing antitumour immunity and chemotherapeutic effect in pancreatic ductal adenocarcinoma

doi: 10.1038/s41416-025-03185-0

Figure Lengend Snippet: a Western blot analysis of C5aR1 knockdown with C5aR1shRNA transfection in KPCY2838, KPCY6419 cells. The expression of C5aR1 in C5aR1shRNA1, 2-transfected cells was decreased by normalisation with β-actin compared to the control cells. b Proliferation assay analysis compared with C5aR1shRNA-transfected cell lines. The absorbance of KPCY2838 and KPCY6419 cells, which had undergone C5aR1 knockdown with C5aR1shRNA1, 2 transfection, showed significant reduction after 48 h in both cells when compared to the control group (* p < 0.05, ** p < 0.01, Mann–Whitney–Wilcoxon test). c Proliferation assay analysis compared with CCX168. The absorbance of KPCY2838 and KPCY6419 cells seeded with CCX168 showed significant reduction after 48 h in KPCY2838 cells; 72 h in KPCY6419 when compared to control group (* p < 0.05, ** p < 0.01, Mann–Whitney–Wilcoxon test). d Proliferation assay analysis compared with Gem and CCX168. Proliferation in KPCY2838 and KPCY6419 cells exposed with CCX168 showed a significant reduction after 48 h in both cells when compared to control group (* p < 0.05, ** p < 0.01, Mann–Whitney–Wilcoxon test). Proliferation in KPCY2838 cells was significantly inhibited after 48 h in the Gem and CCX168 combined group compared to the Gem or CCX168 group. e Experimental design of the in vivo experiment using KPCY2838 cells, which were transfected using two C5aR1shRNAs. f Images of tumours removed after day 19 from orthotopic transplantation using KPCY2838 cells. g The tumour volume was significantly reduced in the C5aR1shRNA 1, 2 groups compared to the control group (** p < 0.01, Mann–Whitney–Wilcoxon test). h – m Images of IHC for C5aR1 in tumour tissues. Compared to the control group, we confirmed that C5aR1 knockdown resulted in a reduction in the expression of C5aR1 in the C5aR1shRNA group. Images of IF staining for DAPI (blue), YFP (green), and C5aR1 (red) expression in tumour tissues. Original magnification: ×200. n – p Images of IHC for cleaved caspase-3 + cells. Original magnification: ×400. q The number of cleaved caspase-3 + cancer cells was significantly higher in the two C5aR1shRNA groups than in the controlshRNA group (** p < 0.01, Mann–Whitney–Wilcoxon test).

Article Snippet: The detailed information of C5a, C5/C5b, C5aR1, and C5aR2 antibodies was indicated as follows; Human C5a mouse monoclonal antibody (D2): cat. no. MA5-43875, Company: Thermo Fisher Scientific Japan, Degree of dilution for IHC and IF: 1:100; Human C5/C5b monoclonal antibody: cat. no. HA255621 , Company: ABclonal, Degree of dilution for IHC: 1:100; Human C5aR1 rabbit polyclonal antibody: cat. no. A1900, Company: ABclonal Hubei China, Cross-reactivity: mouse, rat, Degree of dilution for IHC and IF: 1:150; Human C5aR2 rabbit polyclonal antibody: cat. no. HPA016629, Company: Merck Ltd./Sigma-Aldrich Japan G.K., Degree of dilution for IHC and IF: 1:75.

Techniques: Western Blot, Knockdown, Transfection, Expressing, Control, Proliferation Assay, MANN-WHITNEY, In Vivo, Transplantation Assay, Staining