Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 1. Immunohistochemistry of C5aR in the RCC specimens. FFPE samples of RCC were stained with the anti-C5aR antibody. Representative examples of C5aR-negative RCC without metastasis (A) and C5aR-expressing mRCC (B) are shown. Original, x200 magnification. C5aR, C5a receptor; RCC, renal cell carcinoma; mRCC, metastatic RCC; FFPE, formalin-fixed paraffin-embedded.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Immunohistochemistry, Staining, Expressing, Formalin-fixed Paraffin-Embedded

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 3. C5a elicits cytoskeletal rearrangement and changes in cellular mor- phology in the C5aR-expressing Renca cells. Renca/empty cells (A-D) and Renca/C5aR cells (E-G) were incubated with C5a (10 nM) and fixed at the indicated time-points. F-actin was visualized by immunofluorescent staining with Alexa 488-conjugated phalloidin (green), and nuclei with TO-PRO-3 (red). Scale bars, 20 µm. Arrowheads and the arrow indicate membrane ruffing and lamellipodia, respectively. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Expressing, Incubation, Staining, Membrane

Journal: Oncology reports

Article Title: C5aR is frequently expressed in metastatic renal cell carcinoma and plays a crucial role in cell invasion via the ERK and PI3 kinase pathways.

doi: 10.3892/or.2015.3800

Figure Lengend Snippet: Figure 4. C5a-C5aR axis triggers ERK and PI3K-dependent cellular inva- sion. (A) Renca-derived cells were stimulated with or without 10 nM C5a for 15 min after serum starvation for 24 h. Samples were harvested and subjected to immunoblotting using antibodies indicated. (B and C) Invasion assays were carried out using (B) Renca/empty and Renca/C5aR cells or (C) Renca/C5aR cells only. (C) Medium in both the upper and lower wells were mixed with the indicated kinase inhibitors or vehicle (DMSO). U0126 was used at 20 µM and PI-103 was used at 0.5 µM. Data are presented as the mean ± SD. *P<0.02 and **P<0.01. C5aR, C5a receptor.

Article Snippet: Establishment of C5aR stably expressing Renca cells and in vivo study. pCMV-C5aR that encodes full-length murine C5aR was purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Derivative Assay, Western Blot

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: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing mouse C5aR1-GFP were transfected with 100 nM mouse C5aR1 ON-TARGETplus SMART siRNA–6, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA. 72 h post transfection cells were analyzed by flow cytometry to measure the relative expression of mouse C5aR1-GFP (left panel). CHO cells expressing human C5aR1-GFP were transfected with 100 nM human C5aR1 ON-TARGETplus SMARTpool siRNA, 100 nM GFP siRNA (positive control), or 100 nM negative control siRNA (right panel). Relative knockdown is based on the percentage of the cells that are to the left of the gate relative to the negative control sample. The experiment was carried out twice with similar results.

Article Snippet: The construction and characterization of Chinese hamster ovary (CHO) cells expressing human C5a receptor 1 (C5aR1) and human C5aR1-green fluorescent protein (GFP) have been described previously [ ]. pCMV6 mouse C5aR1 was purchased from OriGene Technologies (catalog number MC208206; GenBank Accession NM_007577).

Techniques: Expressing, Transfection, Positive Control, Negative Control, Flow Cytometry, Knockdown

Journal: PLoS ONE

Article Title: CD177-mediated nanoparticle targeting of human and mouse neutrophils

doi: 10.1371/journal.pone.0200444

Figure Lengend Snippet: CHO cells expressing mouse C5aR1-GFP were transfected with 50 nM or 100 nM LNA GapmeR ASO. The cells were analyzed for C5aR1-GFP expression and mRNA levels 72 h post transfection. A. Relative receptor knockdown was measured by flow cytometry. The percentage knockdown was calculated based on the number of cells to the left of the gate relative to the negative control ASO. B. Relative gene expression was calculated from quantification cycle (Cq) values obtained by RT-qPCR using the ΔΔCq method. To control for possible experimental variation, the qPCR was carried out using two sets of mouse C5aR1 primers (C5aR1 208–402 and 221–430), and two sets of reference primers. The results in the left panel show the relative quantity of C5aR1 mRNA normalized to Eif3i, and the results in the right panel show the relative quantity of C5aR1 mRNA normalized to Vezt. Mock transfected cells received no ASO and non-targeting control (NTC) cells were transfected with a non-targeting ASO. The RT-qPCR was carried out with triplicate samples ± SD. One-way analysis of variance at 95% confidence interval showed that the relative mRNA expression levels were significantly lower in the ASO treated cells compared to the mock transfected and non-targeting ASO cells ( p value <0.0001; ***).

Article Snippet: The construction and characterization of Chinese hamster ovary (CHO) cells expressing human C5a receptor 1 (C5aR1) and human C5aR1-green fluorescent protein (GFP) have been described previously [ ]. pCMV6 mouse C5aR1 was purchased from OriGene Technologies (catalog number MC208206; GenBank Accession NM_007577).

Techniques: Expressing, Transfection, Knockdown, Flow Cytometry, Negative Control, Gene Expression, Quantitative RT-PCR, Control

Journal: PLoS ONE

Article Title: Nanoparticulate matter exposure results in neuroinflammatory changes in the corpus callosum

doi: 10.1371/journal.pone.0206934

Figure Lengend Snippet: (A) Filtered air (n = 8) or nPM (n = 8) exposed mice stained for C5 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (B) Low magnification representation of region analyzed. (C) C5 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.001). (D) Filtered air (n = 8) and nPM (n = 8) exposed mice stained for C5α (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (400x). (E) Low magnification representation of region analyzed. (F) C5α immunostaining in the corpus callosum of nPM exposed animals was significantly greater than in the filtered air group (p = 0.02). (G) Filtered air (n = 18) and nPM (n = 18) exposed mice stained for CD88 (red) in the corpus callosum. Nuclei (DAPI) are stained in blue (200x). (H) Low magnification representation of region analyzed. (I) CD88 immunostaining in the corpus callosum was significantly higher in nPM exposed animals compared to the filtered air group (p = 0.04). * signifies p< 0.05, ** signifies p ≤ 0.001. Error bars represent standard deviation. Scale bars indicate 50 μm.

Article Snippet: Slides were incubated overnight with anti-C5 (mouse 1:50 Hycult Biotech, Netherlands; clone BB5.1), anti-CD88 (rat 1:200 Hycult Biotech, Netherland; HM1076) or rabbit complement component C5α (125kDa) antibody (1:50 Santa Cruz, SC-21941).

Techniques: Staining, Immunostaining, Standard Deviation