Journal: BMC Genomics

Article Title: Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

doi: 10.1186/1471-2164-10-403

Figure Lengend Snippet: Real-time RT-PCR validation of microarray results . The expression of CD16, C3AR1, C1QR1, ICAM-2, CSFR1, CSF3R, CDKN1C, TNFRSF8, and LTB mRNA was quantified by SYBR Green real time RT-PCR in CD16 + and CD16 - Mo. The concentration of each gene was normalized to the 28S rRNA internal control and expressed as fgs RNA of a target gene per 1 ng rRNA28S. Depicted are results (mean ± SD of triplicate wells; *, p < 0.05, unpaired t-test, CD16 + versus CD16 - Mo) obtained with matched cells from 2 different healthy donors.

Article Snippet: Fluorochrome-conjugated Abs used for FACS analysis were CD14, CD16, CD19, CD16b, CD66b, CD56, and CD3 (Beckman Coulter); M-DC8 and CD1c (Miltenyi), HLA-DR, CD114, C3aR, CD1d and CD43 (BD Pharmingen), CD115 (R&D Systems), CD93/C1qR1 (Chemicon International), and CXCL16 (R&D Systems).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Microarray, Expressing, SYBR Green Assay, Concentration Assay, Control

Journal: BMC Genomics

Article Title: Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

doi: 10.1186/1471-2164-10-403

Figure Lengend Snippet: Differential expression of CD114/CSF3R, CD115/CSF1R, CD93/C1qR1 and C3aR1 on CD16 + and CD16 - monocytes . Freshly isolated PBMC were stained with FITC CD14, PE-Cy5 CD16, and PE CD114, PE CD115, and PE CD93 Abs. The expression of CD3aR1 was detected after staining with unconjugated mouse C3AR1 Ab and PE rat anti-mouse Ab (RAM). CD14 high CD16 neg (R2) and CD14 low CD16 + (R3) Mo (A) were analyzed for expression of CD114, CD115, CD93 and C3aR1 (B) . Shown is an overlay histogram from one representative donor of 4 donors examined (B, left panels) and graphs showing mean ± SEM for % or MFI of CD114, CD115, CD93 and C3aR1 expression on each Mo subset (B, right panels) . (*, Paired t-test p-value < 0.05, CD16 + versus CD16 - Mo; n = 4).

Article Snippet: Fluorochrome-conjugated Abs used for FACS analysis were CD14, CD16, CD19, CD16b, CD66b, CD56, and CD3 (Beckman Coulter); M-DC8 and CD1c (Miltenyi), HLA-DR, CD114, C3aR, CD1d and CD43 (BD Pharmingen), CD115 (R&D Systems), CD93/C1qR1 (Chemicon International), and CXCL16 (R&D Systems).

Techniques: Quantitative Proteomics, Isolation, Staining, Expressing

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3aR ameliorates rotenone-induced neurodegeneration, synaptic loss, α-synuclein phosphorylation and cognitive deficits in mice. C57BL/6 mice were treated with rotenone for 3 weeks to establish PD models, with 30 min of SB290157 (C3aR inhibitor, 1 mg/kg, i.p.) administration after each rotenone injection. (A) Concentrations of C3a in the hippocampus of Con and Rot mice, measured by enzyme-linked immunosorbent assay (ELISA). n = 6. (B) Representative Western blot images and densitometric quantification of C3aR protein levels in the hippocampus and rotenone mice. n = 4. (C) The mRNA levels of C3aR in vehicle and rotenone mice. (D) Representative images of double-immunofluorescence staining with C3aR and Iba-1 or Neu-N antibodies and (E, F) the quantification of C3aR + Iba-1 + and C3aR + Neu-N + cells in vehicle and rotenone mice. n = 3. (G) The quantification of Neu-N + cell number and (H) optical density of PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. (I) The representative images of Neu-N and PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. n = 6. (J, K) Representative bands of Neu-N, PSD95 and ser129-phosphorylated α-synuclein and the quantification of blots in rotenone mice with or without C3aR inhibitor treatment. n = 4. (L) Representative images of ser129-phosphorylated α-synuclein and (M) the quantification of staining density in rotenone mice with or without C3aR inhibitor treatment. n = 3. (N–Q) Escape latency, traveled distance, first platform crossing latency and platform crossing number in rotenone mice with or without C3aR inhibitor treatment. n = 15; ∗ p < 0.05, ∗∗ p < 0.01 for comparison between Rot & Rot + C3aR inhibitor groups; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Double Immunofluorescence Staining, Staining, Comparison

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis suppresses microglia-mediated neuroinflammation in rotenone-treated mice. WT mice, C3 −/− mice, and C3aR inhibitor (SB290157)-treated mice were exposed to rotenone for 3 weeks to induce PD-related neuroinflammation. (A) Representative images of Iba-1 immunostaining and (B) the quantification of staining density in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) The mRNA levels of iNOS, IL-1β and TNFα in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (D) Representative bands of p-MAPK, MAPK, p-p65, p65, p-IκBα and IκBα and the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Immunostaining, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis mitigates microglia-mediated abnormal synaptic pruning in rotenone-treated mice. WT mice, C3 −/− mice, and SB290157-treated mice were subjected to rotenone for 3 weeks. (A) Representative images of double fluorescence staining with anti-Iba-1 and anti-CD68 antibodies and (B) the quantification of staining in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) Representative bands of PSD95, p-CREB, CREB, mBDNF and TrkB and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Fluorescence, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis attenuates rotenone-induced activation of dark microglia and related secretion of toxic lipids in vivo and in vitro. (A) Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and (B) the quantification of blots in mice. n = 4. (C) Representative images of double-immunofluorescence staining with p-eIF2α and Iba-1 antibodies in mice. n = 3. (D) BV2 microglial cells were treated with C3 protein (with or without SB290157 or NOX2 inhibitor GSK2795039) for 24 h. Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and the quantification of blots in BV2 cells were shown. (E) The mRNA levels of atf4 and asns in BV2 cells. n = 3. (F) 3D principal component analysis (PCA) score plot showing separation of lipid profiles. (G) The heatmap shows the differential lipid metabolites among groups. (H) Venn diagram showing the overlap of differential lipid metabolites. (I) Classification of differential lipid metabolites at the major category level between C3_C3aR vs. C3 and C3 vs. Control groups. (J) Subclass distribution of differential lipid metabolites between C3_C3aR vs. C3 and C3 vs. Control groups. n = 5. SP: sphingolipids; GL: glycerolipids; FA: fatty acyls; DL: derivatized lipids (biotinylation, diazomethane). ST: sterol lipids. PR: prenol lipids. GP: glycerophospholipids. ∗∗ p < 0.01; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay, In Vivo, In Vitro, Double Immunofluorescence Staining, Control

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Blocking C3/C3aR axis suppresses rotenone-induced PANoptosis in mice. (A) Representative images of TUNEL staining and (B) the quantification of TUNEL + cells in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (C) Representative bands of Bcl-2, Bax, active-caspase-3, p-MLKL, MLKL, p-RIP1, RIP1, p-RIP3, RIP3, NLRP3, caspase-1 and GSDMD and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, TUNEL Assay, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: C3–C3aR axis promotes PANoptosome formation and PANoptosis through z-DNA-ZBP-1 interaction via mitochondrial ROS. SH-SY5Y neuronal cells were treated with different concentrations of C3 protein (with or without SB290157, apoptosis inhibitor Z-VAD, necroptosis inhibitor Nec-1, inflammasome inhibitor MCC950, ROS scavenger NAC, NADPH oxidase inhibitor apocynin, mtROS scavenger mito-TEMPO, z-DNA inhibitor CQ, or z-DNA promoter CeCl 3 ) for 24 h. (A) The survival rate of cells treated with different concentrations of C3 protein. (B) The survival rate of cells treated with C3 with or without different inhibitors. (C) Representative pictures of ZBP-1, AIM2, RIP3 and caspase-1 interaction using COIP. (D) The ratio of A260/A295 absorbance. (E) Representative images of DHE staining and quantification of DHE density in SH-SY5Y cell among groups. (F) Representative images of z-DNA fluorescence staining and quantification of density in SH-SY5Y cell among groups. (G) Representative blots of z-DNA and ZBP-1 and quantification of blot density in SH-SY5Y cell among groups. (H) Representative images of double-immunofluorescence staining using antibodies against z-DNA and ZBP-1 and quantification of ration of z-DNA + ZBP-1 + /ZBP-1 + cells in SH-SY5Y cell among groups. (I) Representative blots of z-DNA and ZBP-1 interaction using COIP. (K) The survival rate of cells treated with C3 with or without apocynin, NAC, mito-TEMPO, CQ and CeCL 3 . n = 3; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm in (E) and 25 μm in (F) and (H).

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Fluorescence, Double Immunofluorescence Staining

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: ( A ) Schematic diagram showing the complement classical pathway function in synapse removal and neuroinflammation via C3b and C3a (created with BioRender.com ). C1 complex (C1q, C1r, and C1s) activation and binding to a synapse leads to deposition of C4b and C2b fragments (C3 convertase), resulting in C3b deposition which is recognized by CR3 on microglia, and release of soluble C3a, recognized by C3aR on microglia. ( B ) Study design showing mouse genotypes and timing of experimental measurements: MRI, immunohistochemistry, and behavior. ( C ) Representative MRI images in 6-month-old (top) and 9-month-old (bottom) male mice with genotypes as indicated. Red arrows in 9-month-old TauP301S mouse images indicate enlargement of ventricles, and white arrows indicate hippocampal atrophy. ( D) Longitudinal MRI quantification of volume changes of ventricles, neocortex, and hippocampus at 6 and 9 months compared with 3 months of age in mice with indicated genotypes. ( E ) Longitudinal MRI quantification of ventricle, neocortex, and hippocampus volumes after first normalizing to total brain volume, showing changes of normalized volumes at 6 and 9 months compared with 3 months mice with indicated genotypes. n = 9-12 male mice per genotype. Data are represented by mean +/- SEM. *, p < 0.05, **, p < 0.01, ***, p < 0.001, two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: Activation Assay, Binding Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: Single cell RNA-sequencing was performed on brain tissues from wild-type (C3aR +/+) and C3aR knockout (C3aR -/-) mice treated with lipopolysaccharide (LPS) or vehicle (control). (A) UMAP plot displaying the clustering of single cells across both genotypes and treatment conditions, color-coded by the major cell types. (B) UMAP plot overlaid with C3ar1 expression (log-normalized counts), demonstrating that C3ar1 expression is restricted to microglia and PVMs. (C) UMAP plot highlighting the C3aR +/+ and C3aR -/- cells derived from animals treated with LPS. (D) Bar plot quantifying the number of differentially expressed genes (DEGs) across all conditions, separated by cell type. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR WT microglia. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (F) Four-way comparison of DE genes in LPS treated microglia in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis. (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x. (G) Volcano plot showing the differential gene expression between C3aR WT and KO microglia in LPS treated mice. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in C3aR KO and blue for downregulated in C3aR KO. (H) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO PVM. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (I) Four-way comparison of DE genes in LPS treated microglia in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis. (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x. (J) Volcano plot showing the differential gene expression between C3aR WT and KO PVM in LPS treated mice. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in C3aR KO and blue for downregulated in C3aR KO.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Knock-Out, Control, Expressing, Derivative Assay, Gene Expression, Comparison, Quantitative Proteomics

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of microglia into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Average expression of genes representing specific microglial states calculated across the dataset and represented in the form of a feature plot. (C) Proportion of each microglial subpopulation across treatment and genotype groups expressed as a percentage of total microglia, illustrating a shift toward activated microglia in response to LPS treatment, independent of C3aR genotype. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (D) Dotplot of the top marker genes of each microglial subpopulation highlighting the transcriptional states associated with activated and proliferative states. (E) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes microglial subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Expressing, Marker

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of astrocytes into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Dotplot of the top marker genes of each astrocyte subpopulation highlighting the transcriptional states associated with LPS treatment. (C) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes astrocyte subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value. (D) Proportion of each astrocyte subpopulation across treatment and genotype groups expressed as a percentage of total astrocytes, illustrating a shift toward an inflammatory state in response to LPS treatment, independent of C3aR genotype. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR WT astrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (F) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO astrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment. (G) Four-way comparison of DE genes in LPS treated astrocytes in C3aR +/+ (x-axis) vs. C3aR -/-genotype (y- axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Marker, Gene Expression, Comparison, Quantitative Proteomics

Journal: bioRxiv

Article Title: Complement C3aR deletion does not attenuate neurodegeneration in a tauopathy model or alter acute inflammation-induced gene expression changes in the brain

doi: 10.1101/2025.06.16.660026

Figure Lengend Snippet: (A) UMAP plot displaying the sub-clustering of oligodendrocyte lineage cells into transcriptionally distinct subpopulations, identified based on single cell RNA-seq analysis. (B) Proportion of each oligodendrocyte lineage subpopulation across treatment and genotype groups expressed as a percentage of total oligodendrocyte lineage cells, illustrating a shift toward an inflammatory state in response to LPS treatment, independent of C3aR genotype. Only cell populations with proportions that are greater than 1% of the total OL lineage cells are shown although all cells were included in the calculation. Differential abundance statistics * FDR < 0.05; ** FDR <0.01 and *** FDR< 0.001. (C) Dotplot of the top marker genes of each astrocyte subpopulation highlighting the transcriptional states associated with LPS treatment. (D) Bubble plot representing the results of a Gene Ontology (GO) term enrichment analysis using EnrichGO. X-axis denotes astrocyte subclusters and y-axis denotes enriched GO terms. The size of the bubble reflects the number of genes contributing to enrichment and the color represents the adjusted p-value. (E) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3arR WT oligodendrocytes. Genes with significant changes (FDR < 0.05) are marked with red for upregulated in LPS and blue for downregulated in LPS treatment (F) Volcano plot showing the differential gene expression between LPS-treated and vehicle-treated C3aR KO oligodendrocytes. (G) Four-way comparison of DE genes in LPS treated oligodendrocytes in C3aR +/+ (x-axis) vs. C3aR -/- genotype (y-axis). Each point represents one gene colored by whether the log Fold change ≥ 2 and FDR ≤ 0.05 in one or both differential expression analysis (red for C3aR +/+, green for C3aR -/-, and blue for both). Corresponding numbers of DE genes are shown near the borders of the plot. Diagonal line, y = x.

Article Snippet: Cells were stained with either Alexa Fluor 647-conjugated isotype control antibody (R&D systems, # IC006R; 1:100) or Alexa Fluor 647-conjugated C3aR antibody (R&D systems, # FAB10417R: 1:100) for 30 min at 4°C and then washed with FACS buffer.

Techniques: RNA Sequencing, Marker, Gene Expression, Comparison, Quantitative Proteomics

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) C3 (A) and C3aR1 (B) mRNA levels in the parahippocampal gyrus of NCI (n=32), MCI (n=34), or AD (Dementia, n=160). Spearman’s correlation, ρ (C3)=0.36, p<2.6E-08; ρ (C3aR)=0.33, p<3.4E-07.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A)Representative GFAP and Iba1 immunostainings with DAPI in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative Western blots of PHF1- and CP13-positive phospho-tau and total tau in PS19 and PS19/C3aR KO mouse brains at 9 months. γ-tubulin is used as a loading control.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Western Blot

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative synaptophysin (Syp) and Syp/NeuN co-immunostaining in area CA3 of hippocampus of WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Immunostaining

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Unsupervised clustering gene expression heatmap from RNAseq analysis of hippocampal tissues of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. 1726 DEGs were identified by comparing the PS19 vs WT group (1155 genes upregulated, red, and 571 downregulated, blue). n=4-5/genotype, adjusted p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Expressing

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Gene-set enrichment analyses (GSEA) of the microglia sensome gene list in PS19 vs WT (left, NES=2.81, FDR<0.001) and in PS19/C3aR KO vs PS19 (right, NES= −3.0, FDR<0.001) comparisons. NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) Total STAT3 (A) and Y705-phospho-STAT3 (B) levels in 9 month-old WT (n=10), C3aR KO (KO, n=10), PS19 (PS, n=7), and PS19/C3aR KO (PS/KO, n=7) mice measured by RPPA (mean ± s.e.m). The asterisks above PS19 are for PS19 vs WT comparison; those above PS19/C3aR KO are for PS19/C3aR KO vs PS19 comparison. Student｀s t-test. *p<0.05; **p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Computational and Structural Biotechnology Journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Complement C3a is highly expressed and is associated with neoadjuvant chemotherapy resistance in pancreatic cancer. (A) Venn diagram, including 3 parts of data: (1) differential genes associated with distinct gemcitabine response (in purple color); (2) differential genes associated with distinct immune infiltration (in blue color); (3) differential genes associated with distinct IC50 value of gemcitabine (in gray color). C3aR1 was the only differential gene shared by all these three parts. (B) Analysis of clinical samples from TCGA(PAAD) database showed that high expression of C3aR1 was associated with poor survival in pancreatic cancer. (C) The boxplot showing the differential expression of C3aR1 in pancreatic cancer tissue (T) and normal pancreatic tissue (N). (D) Representative IHC staining images showed the differential expression of C3, C3a, and C3aR in normal pancreatic tissue and human pancreatic cancer. (E) Representative images of H&E staining and IHC staining showed the differential expression of C3a, and C3aR in normal pancreatic tissue from wild-type (WT) C57BL/6 J mice and in pancreatic cancer tissue from KPC mice. (F) C3a protein levels in human pancreatic cancer tissue and adjacent normal pancreatic tissue were accessed by ELISA. (G) Representative IHC staining images of high/low C3aR expression in pancreatic cancer tissue from FUSCC-TMA. (H) Kaplan–Meier survival analysis of overall survival between high and low C3aR groups in FUSCC-TMA samples. (I) Representative IHC staining images showed the differential expression of C3a and C3aR in pancreatic cancer tissue from gemcitabine-sensitive (GS) patients and gemcitabine-resistant patients. (J) C3a protein levels in pancreatic cancer tissue from GS/GR patients were accessed by ELISA. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative Proteomics, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: The activation of C3a/C3aR pathway promotes the proliferation, migration and gemcitabine resistance in pancreatic cancer cells. (A) Western blotting was used to detect the C3aR expression in Panc-1 cells treated with PBS or 500 ng/ml recombinant C3a protein. (B) Representative images of EdU assays showed that C3a recombinant protein promotes the proliferation of Panc-1 cells in a concentration-dependent manner. (C) The bar plot showed the percentage of EdU + cells per field. (D) Representative images of Transwell assays showed that C3a recombinant protein promotes the migration of Panc-1 cells in a concentration-dependent manner. (E) The bar plot showed the percentage of migrated cells per field. (F) CCK-8 assay showed that C3a recombinant protein increased the IC50 values of Panc-1 cells to gemcitabine. (G) Western blotting was used to detect the C3aR expression in Panc-02 cells treated with PBS or 500 ng/ml recombinant C3a protein (mouse). (H) Representative images of EdU assays showing that C3a recombinant protein (mouse) promotes the proliferation of Panc-02 cells in a concentration-dependent manner. (I) The bar plot showed the percentage of EdU + cells per field. (J) Representative images of Transwell assays showing that C3a recombinant protein (mouse) promotes the migration of Panc-02 cells in a concentration-dependent manner. (K) The bar plot showed the percentage of migrated cells per field. (L) CCK-8 assay showed that C3a recombinant protein (mouse) increased the IC50 values of Panc-02 cells to gemcitabine. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Migration, Western Blot, Expressing, Recombinant, Concentration Assay, CCK-8 Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Knockdown of C3aR does not affect the progression of pancreatic cancer cells in vitro. A.A-F Panc-1 cells were infected with C3aR shRNA to knock down the C3aR expression (sh#1, sh#2), or infected with a scrambled shRNA as the control group (sh-Scr). (A) Western blotting was used to detect C3aR expression in control cells and C3aR-deficient cells to evaluate the transfection efficiency. (B) CCK8 assay was used to evaluate the cell proliferation, and the relative optical density of each group at day 0, 1, 2, 3 and 4 was measured. The line chart showed that C3aR knockdown did not change the proliferation capacity of Panc-1 cells. (C) Representative images of EdU assays showed that the knockdown of C3aR did not affect the proliferation capacity of Panc-1 cells. (D) The bar plot showed the percentage of EdU + cells per field. (E) Representative images of Transwell assays showed that the knockdown of C3aR did not affect the migration ability of Panc-1 cells. (F) The bar plot showed the percentage of migrated cells per field. G-K C3aR-deficient Panc-1cells or control cells were treated with C3a recombinant protein at a concentration of 0 or 500 ng/ml. (G) RT-qPCR was used to detect the C3aR expression in control cells and C3aR-deficient cells with or without C3a treatment. (H) Representative images of EdU assays showed that the proliferation ability of control cells enhanced under the C3a treatment, while that of C3aR-deficient Panc-1 cells did not change. (I) The bar plot showed the percentage of EdU + cells per field. (J) Representative images of Transwell assays showed that the migration ability of control cells enhanced under the C3a treatment, while that of C3aR-deficient Panc-1 cells did not change. (K) The bar plot showed the percentage of migrated cells per field. L-O Ten female BALB/c-nu mice were randomly divided into 2 groups and respectively injected with control Panc-1 cells and C3aR-deficient cells to construct subcutaneous tumor model. Tumor volume was observed and recorded every 3 days. (L) Line charts of volume changes of subcutaneous tumor. (M) On the 21st of treatment, subcutaneous tumors were separated to show tumor size. (N) Western blotting was used to detect the expression of C3aR in subcutaneous tumors. (O) Elisa was used to detect the expression of complement C3a in subcutaneous tumors. (P) Frozen sections were prepared from tumor tissues. IF staining with antibodies to Ki-67 (in red color) was performed to detect proliferation of Panc-1 cells, and DAPI was used to counterstain the cell nuclei (in blue color). (Q) The bar plot showed the percentage of Ki-67 + cells per field.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Knockdown, In Vitro, Infection, shRNA, Expressing, Control, Western Blot, Transfection, CCK-8 Assay, Migration, Recombinant, Concentration Assay, Quantitative RT-PCR, Injection, Construct, Enzyme-linked Immunosorbent Assay, Staining

Journal: Computational and Structural Biotechnology Journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: C3aR antagonist (SB290157) attenuated the effects of C3a activation in pancreatic cancer. (A) Representative images of EdU assays showed that the treatment of SB290157 attenuated C3a-induced proliferation in Panc-1 cells. (B) The bar plot showed the percentage of EdU + cells per field. (C) Representative images of Transwell assays showed that the treatment of SB290157 attenuated C3a-induced migration in Panc-1 cells. (D) The bar plot showed the percentage of migrated cells per field. (E) CCK-8 assay showed that the treatment of SB290157 attenuated C3a-induced gemcitabine resistance in Panc-1 cells. F-K A mouse subcutaneous tumor model of Panc-1 cells was constructed. The mice were randomly grouped and treated with vehicle, 20 mg/kg gemcitabine, 20 mg/kg SB290157, 20 mg/kg gemcitabine combined with 20 mg/kg SB29015 by intraperitoneal injection once a day. (F) Line charts of volume changes of subcutaneous tumor. (G) On the 14th day of treatment, subcutaneous tumors were separated to show tumor size. Frozen sections were prepared from tumor tissues. IF staining with antibodies to Ki-67 (in red color, (H)) and CC3 (in red color, (J)) was performed to detect proliferation and apoptosis of Panc-1 cells. And the percentage of Ki-67 + cells (I) or CC3 + cells (K) per field was shown by bar plots. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Migration, CCK-8 Assay, Construct, Injection, Staining