c5ar1  (Proteintech)

Journal: bioRxiv

Article Title: Consumption of processed foods impairs memory function through dietary advanced glycation end-products

doi: 10.64898/2026.01.07.698065

Figure Lengend Snippet: a , Illustration of the electrophysiology field potential recording setup. b , Ratio of field to volley from electrophysiology field potential recordings of dorsal hippocampus (HPCd) brain tissue slices in rats after 30-d early life AGE-rich diet (n=14 CTL tissue slices, n=15 AGE-rich tissue slices; 2-way repeated measures ANOVA with factors of diet group, fiber volley amplitude [repeated measure], and diet group × fiber volley interaction; P=0.04 diet group, P<0.0001 fiber volley amplitude, P=0.99 diet group × fiber volley amplitude). c , mRNA expression of complement component 3 (C3) in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.04). d , mRNA expression of complement component 5 (C5) in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.68). e , mRNA expression of C3aR in the HPCd (n=7 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.17). f , mRNA expression of C5aR1 in the HPCd (n=8 CTL, n=7 AGE-rich; unpaired two-tailed t-test, P=0.16). g , Structures of the peptide required for complement system receptor activation and of AGEs elevated in the heat-treated diet. h , C5aR1 pERK 1/2 signaling in response to MG-H1, CML, and CEL (smoothed lines indicate nonlinear least squares fit for each treatment). i , C5aR1 Receptor-G protein-Bioluminescence Resonance Energy Transfer (RG-BRET) assay for binding affinities between C5a and MG-H1, CML, CEL, or the C5aR1-selective antagonist avacopan (smoothed lines indicate nonlinear least squares fit for each treatment). Error bars represent standard error of the mean (SEM). *P<0.05. All n’s indicate number of rats or slices per group. Additional details about the statistical analyses for each subpanel can be found in . AGE, advanced glycation end-product; ANOVA, analysis of variance; C3, complement component 3; C3aR, receptor for complement component 3a; C5, complement component 5; C5aR1, receptor for complement component 5a; CA1, Cornu Ammonis subfield 1; CA3, Cornu Ammonis subfield 3; EC, entorhinal cortex; CEL, carboxyethyllysine; CML, carboxymethyllysine; CTL, control [diet]; HPCd, dorsal hippocampus; MG-H1, methylglyoxal-derived hydroimidazolone; pERK, phospho-extracellular signal-regulated kinase; PN, postnatal day; RG-BRET, Receptor-G protein-Bioluminescence Resonance Energy Transfer.

Article Snippet: The following Applied Biosystems probes for use with rats were used: C3 (Rn00566466_m1), C5 (Rn01436156_m1), C3aR (Rn00583199_m1), C5aR1 (Rn02134203_s1), RAGE (Rn01525753_g1), NfĸB (Rn01502266_m1), PSD-95 (Rn00571479_m1), occludin (Rn00580064_m1), claudin-1 (Rn00581740_m1), claudin-5 (Rn01753146_s1), and mucin 2 (Rn01498206_m1). qPCR was performed using Applied Biosystems TaqMan Fast Advanced Master Mix (Cat. no. 444557, ThermoFisher Scientific) and an Applied Biosystems QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific).

Techniques: Expressing, Two Tailed Test, Activation Assay, Bioluminescence Resonance Energy Transfer, Binding Assay, Control, Derivative Assay

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to selective serotonin reuptake inhibitor (SSRI)-induced gene changes. ( B ) GSEA of hepatocellular carcinoma (HCC) RNA-seq data (TCGA cohort) with the SSRI-related gene signature. Sample grouping was made based on the median expression of C5aR1. ( C ) Representative immunohistochemical images showed the expression pattern and cellular distribution of C5aR1 in human HCC tissues. Scale bar, 50 μm. ( D ) Single-cell RNA sequencing analysis showed the expression pattern of C5aR1 with the immune microenvironment of HCC. ( E ) Co-immunofluorescence of C5aR1 (green) with CD163 (red) in HCC samples. Scale bar, 10 μm. ( F ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence and absence of 100 μM citalopram treatment. ( G ) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of citalopram treatment. ( H ) The overall conformation of citalopram binding to C5aR1. ( I ) Representative models of citalopram in pose-1 (left), pose-2 (middle), and allosteric site (right). Several polar interactions were indicated by black dashed lines. ( J ) HEK293T cells were transfected with either WT or mutant C5aR1 expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test ( F ) or one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( G, J ). Figure 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Single Cell, Immunofluorescence, Western Blot, Binding Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of SMART-seq2-based single CD45 + cells. The tSNE (by cluster) was acquired from http://cancer-pku.cn:3838/HCC/ . ( B ) The UMAP showing C5AR1 expression in HCC immune cell clusters. ( C ) Violin plot showing C5AR1 expression in different immune cell clusters.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of selective serotonin reuptake inhibitors (SSRIs) treatment (0, 1, 10, 50, and 100 μM). ( B ) For C5aR1, the predicted binding energy distribution of the clusters with poses more than 50. ( C ) Sequencing analysis showed the successful generation of six C5aR1 mutants. ( D ) The best-scored complex models of C5aR1 with other four different SSRIs. ( E ) HEK293T cells were transfected with either WT or mutant C5aR1 (D282A) expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( E ). Figure 2—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Binding Assay, Sequencing, Transfection, Mutagenesis, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) plot of phagocytosis pathway in macrophages derived from C5ar1 −/− mice and C5ar1 +/− mice. ( B ) Western blotting and immunofluorescence analysis showed C5aR1 protein levels in Cas9-sgControl, -sg C5ar1 THP-1 subclones. ( C ) Effects of C5aR1 deficiency on the macrophage phagocytosis of HCC-LM3 in the presence or absence of C5a stimulation. ( D ) Effects of different selective serotonin reuptake inhibitors (SSRIs) on the macrophage phagocytosis of HCC-LM3 in the presence of C5a stimulation. ( E ) Reconstituted expression of WT and D282A mutant C5aR1 in C5aR1 KO THP-1 cells. ( F ) The effects of citalopram on macrophage phagocytosis in the absence of C5aR1 with reconstituted expression of C5aR1 WT or C5aR1 D282A . In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups. Data are representative of three independent experiments ( C, D, F ). Figure 3—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands. Figure 3—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Expressing, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Conformations of orthosteric binding sites in human (light blue) and mouse (orange) C5aR1. The conformation of human C5aR1 was obtained from the crystal structure (PDB id: 6c1q). The structure of mouse C5aR1 was predicted using the ColabFold (AlphaFold2) software. ( B ) The predicted binding modes of citalopram to human (light blue) and mouse (orange) C5aR1. The conformations of citalopram were shown in pink (binding mode 1) or deep green (binding mode 2) sticks. For mouse C5aR1, green sticks indicate residues set to flexible in the molecular docking process.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Binding Assay, Software

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: Model depicting the molecular mechanism by which citalopram inhibits the Warburg effect and promotes an anti-tumor response in hepatocellular carcinoma (HCC). In the primary HCC microenvironment (left panel), C5aR1-expressing tumor-associated macrophages (TAMs) exhibit reduced phagocytic capacity and an anti-inflammatory state, which correlates with diminished CD8 + T cell anti-tumor immunity and HCC progression. Upon treatment with citalopram (right panel), the drug not only inhibits the glycolytic metabolism of cancer cells by targeting GLUT1 but also acts on C5aR1 expressed by TAMs, thereby enhancing macrophage-driven anti-tumor immunity. Additionally, citalopram induces a systemic immunostimulatory effect on CD8 + T cell functions through yet-to-be-identified serotonergic mechanisms. The dotted line indicates a causal relationship that has not been fully established through direct evidence.

Article Snippet: The following antibodies were used: C5aR1 (1:50, Proteintech, 10375-1-AP) and CD163 (1:200, Abcam, ab182422).

Techniques: Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) identified GLUT1 and C5aR1 as two top hits related to selective serotonin reuptake inhibitor (SSRI)-induced gene changes. ( B ) GSEA of hepatocellular carcinoma (HCC) RNA-seq data (TCGA cohort) with the SSRI-related gene signature. Sample grouping was made based on the median expression of C5aR1. ( C ) Representative immunohistochemical images showed the expression pattern and cellular distribution of C5aR1 in human HCC tissues. Scale bar, 50 μm. ( D ) Single-cell RNA sequencing analysis showed the expression pattern of C5aR1 with the immune microenvironment of HCC. ( E ) Co-immunofluorescence of C5aR1 (green) with CD163 (red) in HCC samples. Scale bar, 10 μm. ( F ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence and absence of 100 μM citalopram treatment. ( G ) The DARTS assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of citalopram treatment. ( H ) The overall conformation of citalopram binding to C5aR1. ( I ) Representative models of citalopram in pose-1 (left), pose-2 (middle), and allosteric site (right). Several polar interactions were indicated by black dashed lines. ( J ) HEK293T cells were transfected with either WT or mutant C5aR1 expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values as mean ± SD and compared by the Student’s t test ( F ) or one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( G, J ). Figure 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: RNA Sequencing, Expressing, Immunohistochemical staining, Single Cell, Immunofluorescence, Western Blot, Binding Assay, Transfection, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of SMART-seq2-based single CD45 + cells. The tSNE (by cluster) was acquired from http://cancer-pku.cn:3838/HCC/ . ( B ) The UMAP showing C5AR1 expression in HCC immune cell clusters. ( C ) Violin plot showing C5AR1 expression in different immune cell clusters.

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) The drug affinity responsive target stability (DARTS) assay and immunoblot analysis showed C5aR1 protein stability against 5 μg/ml pronase in the presence of different concentrations of selective serotonin reuptake inhibitors (SSRIs) treatment (0, 1, 10, 50, and 100 μM). ( B ) For C5aR1, the predicted binding energy distribution of the clusters with poses more than 50. ( C ) Sequencing analysis showed the successful generation of six C5aR1 mutants. ( D ) The best-scored complex models of C5aR1 with other four different SSRIs. ( E ) HEK293T cells were transfected with either WT or mutant C5aR1 (D282A) expression plasmids for 48 hr, followed by DARTS assay with immunoblotting analysis of C5aR1 protein levels. In all panels, *p < 0.05, **p < 0.01. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups ( E ). Figure 2—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Western Blot, Binding Assay, Sequencing, Transfection, Mutagenesis, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Western blotting showed the knockdown efficiency of GLUT1 in mouse Hepa1-6 cells. ( B ) GLUT1 KD Hepa1-6 cells were subcutaneously injected into the Rag1 −/− or immunocompetent C57BL/6 mice, and mice were treated with 5 mg/kg citalopram when bore visible tumors; 3 weeks later, tumor burden was examined ( n = 6–7 per group). ( C ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host ( n = 7). ( D ) Immunofluorescence analysis of C5a deposition in GLUT1 KD Hepa1-6 tumors from C5ar1 +/− and C5ar1 −/− C57BL/6 host. Scale bar, 50 μm. ( E ) Experimental design of bone marrow transfer experiments. ( F, G, I ) GLUT1 KD Hepa1-6 cells were subcutaneously implanted into syngeneic recipient (r) mice that had been reconstituted with bone marrow cells from either C5ar1 +/− or C5ar1 −/− donor mice. The therapeutic effect of citalopram ( F ), C5a deposition ( G ), and macrophage phagocytosis ( I ) in this model was analyzed. Scale bar, 50 μm. ( H ) The phagocytic capacity of macrophages isolated from GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host. Flow cytometry showed the infiltration of CD45 + CD11b + F4/80 + macrophages ( J ), CD206 + TAMs and CD11b + TAMs ( K ), tumor-infiltrating lymphocytes ( L ) in tumor tissues from orthotopic xenograft model, which was generated in immunocompetent C57BL/6 mice with Hepa1-6 cells ( n = 5 per group). ( M, N ) Measurement of CD8 + T cell function in tumor tissues from the groups mentioned in C and F . ( O ) The growth kinetics of GLUT1 KD Hepa1-6 tumors in C5ar1 +/− and C5ar1 −/− C57BL/6 host upon CD8 + T cell depletion ( n = 7). ( P ) Correlation analysis of C5aR1 expression and immune checkpoint molecules, gene signatures of TAMs, exhausted T cells, and effector Tregs in the TCGA cohort ( n = 371). In all panels, *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. Values are presented as mean ± SD and compared by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons ( B, C, F, O ), Student’s t test ( H–M ), one-way ANOVA multiple comparisons with Tukey’s method ( B, N ), and the Spearman’s rank correlation methods ( P ). Figure 3—source data 1. Original western blots for , indicating the relevant bands. Figure 3—source data 2. Original files for western blot analysis displayed in .

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Western Blot, Knockdown, Injection, Immunofluorescence, Isolation, Flow Cytometry, Generated, Cell Function Assay, Expressing

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Gene Set Enrichment Analysis (GSEA) plot of phagocytosis pathway in macrophages derived from C5ar1 −/− mice and C5ar1 +/− mice. ( B ) Western blotting and immunofluorescence analysis showed C5aR1 protein levels in Cas9-sgControl, -sg C5ar1 THP-1 subclones. ( C ) Effects of C5aR1 deficiency on the macrophage phagocytosis of HCC-LM3 in the presence or absence of C5a stimulation. ( D ) Effects of different selective serotonin reuptake inhibitors (SSRIs) on the macrophage phagocytosis of HCC-LM3 in the presence of C5a stimulation. ( E ) Reconstituted expression of WT and D282A mutant C5aR1 in C5aR1 KO THP-1 cells. ( F ) The effects of citalopram on macrophage phagocytosis in the absence of C5aR1 with reconstituted expression of C5aR1 WT or C5aR1 D282A . In all panels, *p < 0.05, **p < 0.01, ***p < 0.001. Values are presented as mean ± SD and compared by one-way analysis of variance (ANOVA) multiple comparisons with Tukey’s method among groups. Data are representative of three independent experiments ( C, D, F ). Figure 3—figure supplement 2—source data 1. Original western blots for , indicating the relevant bands. Figure 3—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Derivative Assay, Western Blot, Immunofluorescence, Expressing, Mutagenesis

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: ( A ) Conformations of orthosteric binding sites in human (light blue) and mouse (orange) C5aR1. The conformation of human C5aR1 was obtained from the crystal structure (PDB id: 6c1q). The structure of mouse C5aR1 was predicted using the ColabFold (AlphaFold2) software. ( B ) The predicted binding modes of citalopram to human (light blue) and mouse (orange) C5aR1. The conformations of citalopram were shown in pink (binding mode 1) or deep green (binding mode 2) sticks. For mouse C5aR1, green sticks indicate residues set to flexible in the molecular docking process.

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Binding Assay, Software

Journal: eLife

Article Title: Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1 + TAMs

doi: 10.7554/eLife.103016

Figure Lengend Snippet: Model depicting the molecular mechanism by which citalopram inhibits the Warburg effect and promotes an anti-tumor response in hepatocellular carcinoma (HCC). In the primary HCC microenvironment (left panel), C5aR1-expressing tumor-associated macrophages (TAMs) exhibit reduced phagocytic capacity and an anti-inflammatory state, which correlates with diminished CD8 + T cell anti-tumor immunity and HCC progression. Upon treatment with citalopram (right panel), the drug not only inhibits the glycolytic metabolism of cancer cells by targeting GLUT1 but also acts on C5aR1 expressed by TAMs, thereby enhancing macrophage-driven anti-tumor immunity. Additionally, citalopram induces a systemic immunostimulatory effect on CD8 + T cell functions through yet-to-be-identified serotonergic mechanisms. The dotted line indicates a causal relationship that has not been fully established through direct evidence.

Article Snippet: For the C5aR1 knockdown study, C57BL/6J- C5ar1 em1Cya mice were purchased from Cyagen Biosciences (S-KO-01274, Suzhou, China).

Techniques: Expressing

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