Journal: Cell systems

Article Title: Pharmacoproteomics identifies kinase pathways that drive the epithelial-mesenchymal transition and drug resistance in hepatocellular carcinoma

doi: 10.1016/j.cels.2020.07.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: C3A, <10 Passages , ATCC , Cat#: CRL-10741.

Techniques: Virus, Recombinant, Mass Spectrometry, High Throughput Screening Assay, shRNA, Software, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a bind to distinct types of DNA. (A–C) Gel shift assays presenting interaction between C3(a) and genomic DNA isolated from Raji cells. Isolated genomic DNA was incubated either with pre-titrated concentrations of C3 (A) or C3a (B) or C3b (C) and separated by agarose gel electrophoresis. Formation of high molecular weight DNA-protein complexes is indicated by slower DNA migration (DNA shift) compared to migration of the free nucleic acid. The presence of C3 and its cleavage fragments in the pre-formed protein-DNA complexes was confirmed with anti-C3a and anti-C3d antibodies caused supershift. One representative experiment out of five performed is shown. (D) Gel shift assays showing that C3(a)—DNA interaction is not ionic in nature. C3 or C3a were incubated with genomic DNA of Raji cells in the presence of increasing NaCl concentration (ranging from 150 to 1,200 mM). Binding of C3 and C3a to DNA was observed at 8X higher salt concentration than the physiological 150 mM. (E) Interaction of C3 and C3a with DNA is not restricted to genomic DNA. Linearized pCEP4 vector was incubated with either C3 or C3a and DNA-protein complex formation was investigated by gel shift assay. Results illustrated are one representative out of three independent experiments. (F,G) ELISA results confirming interaction between C3(a) and DNA. ELISA microplate surfaces were coated either with DNA (F) or C3, C3a, C3b (G) . After incubation with distinct concentrations of C3 proteins (F) or Raji genomic DNA (G) , protein-DNA interactions were detected using anti-C3d, anti-C3a (anti-C3 ELISA, F ), or anti-dsDNA antibody (anti-DNA ELISA, G ). Results shown are mean absorbance values measured at 490 nm with SD of four independent experiments. (H) ELISA results showing presence of C3-DNA complexes in formaldehyde cross-linked chromatin fraction of Raji B cells. Cells were incubated in the presence or absence of C3, C3met, fixed with 1% formaldehyde to cross-link protein-DNA complexes and chromatin isolated using commercial kit. C3-DNA complexes were investigated by ELISA on anti-C3 or anti-DNA coated plates. Data are shown as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to non-treated cells (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01).

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Electrophoretic Mobility Shift Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Migration, Concentration Assay, Binding Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Labeling

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: All inductions were performed for 3 days with 1 ×10 6 CD62L hi Foxp3 − CD25 − CD4 + T cells, rhIL-2 (5 ng/ml), and anti-CD3 + CD28 beads (per manufacturer’s instructions), experiments were repeated two times. ( a ) WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 − cells were activated and assayed for percent Foxp3 + CD25 + by flow cytometry (*P<0.001, n=5). (b) Following iT reg induction (as in a ), flow sorted WT, C3ar1 −/− , C5ar1 −/− , or C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were incubated with 10 6 CellTracker Red™ labeled CD25 − Foxp3 − CD4 + WT cells in differing T eff /iT reg ratios + anti-CD3 (5 μg/ml) and 2.5×10 5 CD11c + DCs. Relative suppressive capacity was determined by percent Red-labeled dividers (n=5). (c) WT Foxp3 − CD4 + T cells were induced as in (a) in the absence or presence of anti-C3a (10 μg/ml), anti-C5a (10 μg/ml), or both or in the absence and presence of the antagonists C3aR-A (10 nM), C5aR-A (10 nM), or both. CD4 + T cells then were assayed for Foxp3 expression by flow cytometry (*P<0.001, n=6). (d) Following iT reg induction (as in a ), sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 + CD25 + cells were washed and recultured for 24 hr in the absence of further stimulation. Culture supernatants were assayed for TGF-β, IL-6, and IL-10 by ELISA (*P<0.001, n=6). (e) C3ar1 −/− C5ar1 −/− iT regs were induced (as in a) in the absence and presence of anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Foxp3 + CD25 + T reg percentages were quantified by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Flow Cytometry, Incubation, Labeling, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: iT reg induction and activation experiments were performed as in with 2.5×10 5 CD11c + DCs and anti-CD3 mAb (5 μg/ml) instead of anti-CD3+CD28 beads, experiments were repeated two times. (a) Sorted WT and C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with WT DCs ± TGF-β1 (5 ng/ml) and assayed for CD25 and Foxp3 expression by flow cytometry (*P<0.001, n=10). (b) (Left) WT Foxp3 − CD4 + T cells were incubated with C3 −/− C5 −/− DCs without TGF-β1 ± C3a/C5a (100 ng/ml) and Foxp3 + CD25 + cells quantified by flow cytometry. (Right) WT Foxp3 − CD4 + T cells were incubated in the presence of TGF-β1 (5 ng/ml) ± C3a/C5a (100 ng/ml) and percent Foxp3 + CD25 + cells quantified (*P<0.001, n=7). (c) Sorted WT or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with C3ar1 −/− C5ar1 −/− DCs in the absence or presence of C5a (100 ng/ml) after which percent Foxp3 + CD25 + cells was quantified. The % Foxp3 + cells with DKO or WT T cells did not significantly differ (P=0.54). (d) Sorted C3 −/− C5 −/− or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated with Daf1 −/− DCs ± anti-C3a and anti-C5a mAbs after which percent Foxp3 + CD25 + cells was quantified (*P<0.001; n=6). (e) Sorted WT Foxp3 − or C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were incubated for 24 hr either in the absence of DCs or in the presence of WT DCs or C3ar1 −/− C5ar1 −/− DCs. Supernatants then were assayed for TGF-β1 and IL-6 by ELISA (*P<0.001, n=6). (f) C3ar1 −/− C5ar1 −/− Foxp3 − cells were incubated for 3 days with anti-CD3 (5 μg/ml), IL-2 (5 ng/ml), and WT DCs ± anti-TGF-β mAb (5 μg/ml), TGF-βR1 inhibitor (10 nM), or Smad3 inhibitor (5 μM). Following the 3 day induction, Foxp3 + CD25 + T reg percentages were determined by flow cytometry.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Activation Assay, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) WT CD4 + T cells were incubated for 30 min with anti-CD3+CD28 beads after which they were incubated for 10 min with Forskolin (30 μM) ± C3a (100 nM), C5a (100 nM), or both. Levels of cAMP activity were determined by cAMP-Glo assay (*P<0.001). (b) WT, C3ar1 −/− , C5ar1 −/− , and C3ar1 −/− C5ar1 −/− CD4 + T cells were stimulated with anti-CD3 mAb for 30 min after which PKA activity was quantified by PepTag assay. (c) iT regs generated from sorted WT Foxp3 − CD4 + T cells plus TGF-β1, iT regs derived from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells, and conventional Foxp3 − CD4 + T cells were stimulated with anti-CD3+CD28 beads for 15 min and assayed for S 473 and T 308 p-AKT by Luminex assay (n=5). (d and e) WT or C3ar1 −/− C5ar1 −/− CD4 + T cells were incubated for 15 min ± anti-CD3+CD28 beads. Cells were extracted in phospho-lysis buffer and extracts assayed for (d) phospho-rbS6 and (e) phospho-Smad2 by immunoblotting. (f) iT regs induced from WT Foxp3 − CD4 + T cells plus TGF-β1 or from C3ar1 −/− C5ar1 −/− Foxp3 − CD4 + T cells were assayed for phospho-STAT3 and phospho-STAT5 by flow cytometry (representative plots of n=6).

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Activity Assay, Glo Assay, Generated, Derivative Assay, Luminex, Lysis, Western Blot, Flow Cytometry

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Sorted WT Foxp3 − cells were incubated for 1 hr with anti-CD3+CD28 beads (as in ) in the absence or presence of TGF-β1 (5 ng/ml) and then assayed for complement mRNA transcripts by qPCR (*P<0.001, n=5). (b) Sorted WT Foxp3 − CD4 + T cells were incubated for 1 hr with anti-CD3+CD28 beads plus TGF-β1 (5 ng/ml) alone, TGF-β1 (5 ng/ml) plus IL-6 (5 ng/ml), or IL-6 (5 ng/ml) alone, after which the cells were assayed for C3 mRNA expression by qPCR (*P<0.001, **P<0.02, n=5). (c and d) Sorted WT Foxp3 − CD4 + T cells were incubated for 48 hr with anti-CD3+CD28 beads plus TGF-β1 alone, TGF-β1+IL-6, or IL-6 alone as in (b) . Culture supernatants were assayed for (c) C3a and C5a generation by ELISAs, and (d) C3aR and C5aR surface expression by flow cytometry (P<0.01 for TGF-β1 alone vs TGF-β1+IL-6 or IL-6 alone, Mean Fluorescence Intensity (MFI) values; n=5). (e) Sorted WT Foxp3 − cells were activated for 48 hr with anti-CD3+CD28 beads ± C5a (100 ng/ml) or C3aR-A/C5aR-A (10 nM), after which culture supernatants were assayed for TGF-β1 or IL-6 by ELISA (P<0.05; n=6). (f) Sorted WT Foxp3 − CD4 + T cells were incubated with WT DCs or with C3 −/− C5 −/− DCs in the absence of TGF-β1 ( C3 −/− C5 −/− iT regs ). DCs (left side) were assayed for C5aR and C5L2 expression by gating on CD11c + cells. Responder T cells (right side) were assayed for C5aR and C5L2 expression by gating on Foxp3 − cells in the case of WT CD4 + T cells prepared with WT DCs and on Foxp3 + cells in the case of WT CD4 + prepared with C3 −/− C5 −/− DCs. (*P < 0.01; n=5). (g) Sorted WT or C5L2 −/− (encoded by Gpr77) Foxp3 − CD4 + T cells were incubated with WT DCs (Left) and sorted WT Foxp3 − CD4 + T cells were incubated with WT or C5L2 −/− DCs (right) both in the presence of anti-CD3 and TGF-β1 (5 ng/ml). Percent Foxp3 + CD25 + cells were assayed by flow cytometry. (*P<0.001, n=6). (h) WT Foxp3 − CD4 + T cells were incubated for 3 days with WT DCs plus TGF-β1 (5 ng/ml) after which Foxp3 + and Foxp3 − cells were sorted. Foxp3 − CD4 + T cells or Foxp3 + CD4 + iT regs were incubated for 5 min with anti-CD3+CD28 beads + biotin-labeled C5a, the cells chilled to 4°C, and plasma membrane fractions were isolated by ultracentrifugation. Following C5aR and C5L2 IP, bound proteins were eluted with alkaline solution, and equal amounts of protein (determined by A 280 ) were loaded onto SDS PAGE gels. Following electrophoresis, gels were blotted for biotinylated-C5a by adding streptavidin-HRP followed by ECL reagent.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Labeling, Membrane, Isolation, SDS Page, Electrophoresis

Journal: Nature immunology

Article Title: Absent C3a and C5a receptor signaling into CD4 + T cells enables auto-inductive TGF-β1 signaling and induction of Foxp3 + T regulatory cells

doi: 10.1038/ni.2499

Figure Lengend Snippet: Experiments were repeated two times. (a) Flow sorted human CD45RA + CD25 − CD4 + T cells (1×10 6 ) were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous CD11c + DCs in the absence or presence of 1) TGF-β1 (5 ng/ml), 2) each of C3aR-A/C5aR-A (10 nM), or 3) each of anti-C3a/C5a mAbs (10 μg/ml). Percent Foxp3 + CD25 + CD4 + T cells then were determined by flow cytometry. (b) Flow sorted CD25 + cells from (a) were incubated for 3 days with in differing T eff /iT reg ratios with 1×10 6 CFSE labeled autologous naive CD25 − cells, anti-CD3 mAb (3 μg/ml), and 1×10 4 autologous CD11c + DCs. Percent dividers was determined by CFSE dilution. (c) CD45RA + CD25 − CD4 + T cells (1×10 6 ) were isolated from 5 healthy controls (NC) and 3 MS patients by flow sorting. The cells were incubated for 3 days with soluble anti-CD3 mAb (3 μg/ml), rhIL-2 (5 ng/ml), and 2.5×10 5 autologous DCs in the absence or presence of 1) rhTGF-β1 (5 ng/ml), 2) C3aR-A/C5aR-A (10 nM), or 3) anti-C3a/C5a mAbs (5 μg/ml). Cells were washed and sorted on CD25. After sorting, CD25 + (T reg ) were incubated for 3 days with anti-CD3 mAb, 2.5×10 5 autologous DCs, and 10 6 CD25 − (Effector) cells prelabeled with CFSE. Percent dividers was determined by CFSE dilution.

Article Snippet: Anti-Human C3a (20C-CR6032RP) was purchased from Fitzgerald and anti-human C5a (AF2037) was purchased from R&D Systems.

Techniques: Incubation, Flow Cytometry, Labeling, Isolation

Journal: bioRxiv

Article Title: Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

doi: 10.1101/2019.12.12.874511

Figure Lengend Snippet: (A) Normalized enrichment scores of Molecular Signatures Database Hallmark Gene Sets (rows) in each cell type (columns), from Gene Set Enrichment Analysis (GSEA). (B) Expression (fpkm) of three complement-pathway genes ( c3a.1 , c5 , and c9 ) across all three cell types. Each dot represents one replicate.

Article Snippet: Either 3μM Complement Component C3a (R&D Systems #3677-C3-025) or 3μM Complement Component C5a (R&D Systems #2037-C5-025/CF) or 1 μM IL-8 (R&D Systems # 208-IL-10/CF) was loaded onto the devices.

Techniques: Expressing

Journal: bioRxiv

Article Title: Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

doi: 10.1101/2019.12.12.874511

Figure Lengend Snippet: (A) Schematic of c3a.1 locus (top), with exons represented as black boxes. * indicates approximate location of A>T nonsense mutation in exon 22 in sa31241 mutant. Schematic of WT (middle) and mutated (bottom) C3a.1 protein, with selected Pfam- predicted domains noted. Green: α2 macroglobulin; red: thiolester bond-forming region; yellow: α2 macroglobulin complement component. (B) RT-qPCR validation of c3a , c3b , and c5a orthologue expression in pooled WT and c3a.1 −/− (sa31241) whole zebrafish larvae, normalized to WT expression for each gene and to ef1α , with data expressed as mean +/− SEM. Data comprise 3 independent experiments, performed in triplicate, n = 50 larvae per condition per experiment. B, C. 3 dpf WT or c3a.1 −/− zebrafish larvae were subjected to wounding by tail transection (dashed line), subsequently stained with Sudan Black B, and the tail region (box) was imaged. (B) Representative images and (C) quantification of neutrophil recruitment following tail transection in WT (n = 31, 2 hpw; 32, 8 hpw) and c3a.1−/− (n = 41, 2 hpw; 33, 8 hpw) larvae, with data expressed as mean +/− SEM. (D) Quantification of total neutrophil counts in WT (n = 21) and c3a.1 −/− (n = 35) larvae, with data expressed as mean +/− SEM. For C and D, each dot represents one larva; colors represent results of 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001

Article Snippet: Either 3μM Complement Component C3a (R&D Systems #3677-C3-025) or 3μM Complement Component C5a (R&D Systems #2037-C5-025/CF) or 1 μM IL-8 (R&D Systems # 208-IL-10/CF) was loaded onto the devices.

Techniques: Mutagenesis, Quantitative RT-PCR, Biomarker Discovery, Expressing, Staining

Journal: bioRxiv

Article Title: Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

doi: 10.1101/2019.12.12.874511

Figure Lengend Snippet: A, B. WT (n = 71, 1 hpw; 65, 6 hpw) and c3a.1 −/− (n = 71, 1 hpw; 68, 6 hpw) larvae were inoculated with 1000 CFU Pseudomonas aeruginosa in the left otic vesicle and subsequently stained with Sudan Black B. (A) Representative images and (B) quantification of neutrophil recruitment following otic vesicle infection, with data expressed as mean +/− SEM. Each dot represents one larva; colors represent results of 3 independent experiments. ***p<0.001 (C) WT (n = 24) and c3a.1 −/− (n = 32) larvae were infected with 7500 CFU Pseudomonas aeruginosa in the left otic vesicle and survival was tracked over 5 days post-infection. (D) To test whether survival was neutrophil-dependent, c3a.1 +/+ (n = 16) and c3a.1−/− (n = 14) larvae with neutrophils that are mcherry-labeled and carry a mutation in rac2 rendering them migration-defective ( Tg ( mpx:rac2 D57N -mcherry )) were infected with Pseudomonas aeruginosa as in C and survival was tracked over 5 days post-infection. C and D each comprise 3 independent experiments.

Article Snippet: Either 3μM Complement Component C3a (R&D Systems #3677-C3-025) or 3μM Complement Component C5a (R&D Systems #2037-C5-025/CF) or 1 μM IL-8 (R&D Systems # 208-IL-10/CF) was loaded onto the devices.

Techniques: Staining, Infection, Labeling, Mutagenesis, Migration

Journal: bioRxiv

Article Title: Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

doi: 10.1101/2019.12.12.874511

Figure Lengend Snippet: (A)Representative tracks of 5 × 10 6 human neutrophils exposed to a gradient of either C3A or C5A in a microfluidic device. Yellow tracks represent net migration toward source, and red tracks represent net migration away from the source. Black arrow indicates direction of gradient from low concentration to high concentration. (B) Quantification of chemotactic index of human neutrophils exposed to a gradient of either C3A (3 µM source) or C5A (3 µM source), expressed as mean+/−SEM. **p<0.01. (C-D) Human primary neutrophils were pre-treated with 3 µM C3A for 30 min and their migration characteristics toward a gradient of IL-8 (1 uM source), compared with non-pretreated neutrophils. (C) Quantification of total number of neutrophils reaching the chemotactic source in 45 minutes, expressed as mean +/− SEM. (D) Quantification of time required for 20% (left) or 50% (right) of all neutrophils that will reach the chemotactic source over 45 minutes to arrive, expressed as mean +/− SEM. For C and D, each dot represents the result of one technical replicate; colors indicate the results of 3 biologic replicates. *p<0.05, **p<0.01.

Article Snippet: Either 3μM Complement Component C3a (R&D Systems #3677-C3-025) or 3μM Complement Component C5a (R&D Systems #2037-C5-025/CF) or 1 μM IL-8 (R&D Systems # 208-IL-10/CF) was loaded onto the devices.

Techniques: Migration, Concentration Assay

Journal: bioRxiv

Article Title: Cell type specific gene expression profiling reveals a role for the complement component C3A in neutrophil migration to tissue damage

doi: 10.1101/2019.12.12.874511

Figure Lengend Snippet: (A) Time-lapse photomicrographs of neutrophil recruitment to tail-transected caudal fins of c3a.1 +/+ (n= 8) or c3a.1 −/− larvae (n = 8) with mcherry-labeled neutrophils ( Tg(mpx:mcherry)) , 0-60 minutes post-wound, showing tracks of forward migrating neutrophils. (B) Quantification of mean track speed of forward-migrating neutrophils, expressed as mean +/− SEM. Each dot represents the mean of the first 5 neutrophils recruited to the wound of an individual larva. Colors represent the results of 4 independent experiments. *p<0.05 (C) Graph, expressed as mean, and (D) quantification of instantaneous speed of all forward-migrating neutrophils over the first 60 minutes following wounding. In (D), for speed and fold change, data are expressed as median (center values), with 95% confidence intervals (small print). Data comprise 4 independent experiments. *p<0.05.

Article Snippet: Either 3μM Complement Component C3a (R&D Systems #3677-C3-025) or 3μM Complement Component C5a (R&D Systems #2037-C5-025/CF) or 1 μM IL-8 (R&D Systems # 208-IL-10/CF) was loaded onto the devices.

Techniques: Labeling

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A activates C′ by modulating the mRNA and protein expression of the C′ components in vitro. A) Normal human primary AECs were exposed to recombinant human IL-17A (100 ng/ml; 24 h). Subsequent to RNA isolation, cDNA was generated and subjected to real-time quantitative PCR analyses. Means ± sem; Student’s t test. B) Protein lysates from AECs exposed to recombinant human IL-17A (100 ng/ml; 4 h) were subjected to immunoblot analysis against C3aR, C5aR, DAF, and vinculin. C) Conditioned medium collected from AECs exposed to the indicated doses of IL-17A for 4 h was analyzed for active C3a by ELISA. Means ± sem. D) AECs exposed to IL-17A (100 ng/ml; 4 h) were methanol fixed, immunostained for DAF, and counterstained with DAPI. Results represent 3 independent experiments. Scale bars, 100 µm.

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, In Vitro, Recombinant, Isolation, Generated, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis

doi: 10.1096/fj.201700289R

Figure Lengend Snippet: IL-17A deficiency protects against BLEO-induced local C′ activation. The BALF samples collected for the analysis shown in Fig. 2 were also analyzed for C3a (A), C5a (B), and C5b-9 (C) levels by ELISA (n = 7–8/group). Means ± sem. One-way ANOVA, Newman-Keuls (A, C), and Bonferroni (B).

Article Snippet: The antibodies used for immunoblot analysis or immunofluorescent labeling are as follows: decay-accelerating factor (DAF)-clone H-319 (sc-9156); and vinculin-clone7F9 (sc-73614) (Santa Cruz Biotechnology), C3a receptor (C3aR; NBP1-61567), and C5a receptor (C5aR; NBP2-15649; Novus Biologicals, Littleton, CO, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

doi: 10.1186/s12974-022-02516-9

Figure Lengend Snippet: C3a activates astrocytes, promoting MB cell proliferation in vitro. a – d Purified primary MB cells were treated with C3a at the indicated concentration for 48 h in vitro. Immunostaining of Ki67 in red color ( a and b ) or cleaved caspase 3 (CC3, c and d ) in green color was performed to determine the proliferation and apoptosis of MB cells. DAPI-counterstained cell nuclei are shown in blue color. Quantifications of the percentages of Ki67 + cells ( b ) and CC3 + cells ( d ) were plotted according to a and c, respectively. e – f Primary astrocytes were stimulated with C3a at the indicated concentration for 48 h in vitro. Immunostaining of GFAP (green) was performed to examine astrocyte activation ( e ), and the percentage of GFAP + cells was quantified ( f ). Astrocytes were also harvested after C3a administration for GFAP expression assessment by western blotting at the protein level ( g ) and qPCR at the mRNA level ( h ). GAPDH served as a protein sample loading control. ** p < 0.01, *** p < 0.001 vs. (0 nM C3a) group in b , d , f and h . ns: not significant. i , j Astrocytes were pretreated with C3a at the indicated concentration for 24 h and washed thoroughly before being cocultured with MB cells at a 1:10 ratio for another 48 h. Then, the cells were immunostained with Ki67 and GFAP ( i ), and the percentage of Ki67 + /GFAP− cells was quantified ( j ). ** p < 0.01, *** p < 0.001 vs. (MB alone) group in j . Images are representative, and values are expressed as the mean ± SEM from at least three independent experiments

Article Snippet: For detecting C3, anti-human/mouse C3 polyclonal antibody (GeneTex) was used, and for detecting C3a, the neoepitope recognizing antibodies were used as follows: anti-human C3a monoclonal antibody, clone 2991 (Bio-Rad) and anti-mouse C3a monoclonal antibody, clone I87-1162 (BD Biosciences). a Representative IHC staining of intact C3 (left) and C3a (right) in human Hh-type MB tissue; brown color displays the positive staining.

Techniques: In Vitro, Purification, Concentration Assay, Immunostaining, Activation Assay, Expressing, Western Blot, Control

Journal: Journal of Neuroinflammation

Article Title: Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

doi: 10.1186/s12974-022-02516-9

Figure Lengend Snippet: C3a enhances astrocyte activation and MB progression in vivo. A subcutaneous transplantation MB model was established to investigate the function of C3a in MB tumor growth in vivo. Whenever the subcutaneous tumor volume reached 200 mm 3 , tumor-bearing mice were treated with the C3aR antagonist SB290157 or the vehicle control (MCT) daily by intraperitoneal injection (30 mg/kg per mouse per day). Subcutaneous tumor volume was monitored every day, and the volume change was curved based on the fold changes ( a ), * p < 0.05, ** p < 0.01. At the termination of the treatment experiment, tumors were dissociated and photographed to show tumor size ( b ). c – e Frozen sections were prepared with tumor tissues after treatment. Immunostaining of Ki67 (in red, upper panel in c ) and GFAP (in green, lower panel in c ) was performed to determine tumor cell proliferation and TAA activation, and DAPI was used to counterstain the cell nuclei. The percentage of Ki67 + cells ( d ) and the numbers of GFAP + cells per visual field ( e ) in tumor sections were quantified, *** p < 0.001 vs. the MCT treatment group. The results shown are representative of at least two independent experiments. Values are expressed as the mean ± SEM ( n = 4 mice/group)

Article Snippet: For detecting C3, anti-human/mouse C3 polyclonal antibody (GeneTex) was used, and for detecting C3a, the neoepitope recognizing antibodies were used as follows: anti-human C3a monoclonal antibody, clone 2991 (Bio-Rad) and anti-mouse C3a monoclonal antibody, clone I87-1162 (BD Biosciences). a Representative IHC staining of intact C3 (left) and C3a (right) in human Hh-type MB tissue; brown color displays the positive staining.

Techniques: Activation Assay, In Vivo, Transplantation Assay, Control, Injection, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

doi: 10.1186/s12974-022-02516-9

Figure Lengend Snippet: C3a activates astrocytes via the p38 MAPK pathway. a Primary astrocytes were cultured in vitro and stimulated with C3a at the indicated concentrations for 2 and 4 h. Then, the cells were harvested and lysed for total p38 and phosphorylated p38 (p-p38) detection by western blotting. GAPDH served as a protein sample loading control. b , c Astrocytes were administered with C3a at the indicated concentrations in the presence or absence of a p38 inhibitor SB203580 (2 μM) as indicated for 12 h ( b ) or 24 h ( c ). Then, the cells were harvested to perform qPCR for evaluations of IL-6 and TNF-α expression ( b ), or the conditioned medium was collected to assess the secretion of TNF-α protein by ELISA ( c ). d Astrocytes were administered with C3a (100 nM) in the presence or absence of a C3aR antagonist SB290157 (2 μM) for 24 h, then the conditioned medium was collected to assess the TNF-α production by ELISA, ** p < 0.01 vs. C3a (−) & SB290157 (−) group, ## p < 0.01 vs. C3a ( +) & SB290157 (−) group. e – f Astrocytes were pretreated as above and washed thoroughly before coculture with MB cells derived from Math1-Cre/Ptch1 loxp/loxp /eGFP mice at a 1:10 ratio. Forty-eight hours later, the cells were fixed to perform immunostaining for Ki67 (red). DAPI was used to counterstain the cell nuclei ( e ). The percentage of Ki67 + in GFP + cells was quantified ( f ). In b, c and f, * p < 0.05, *** p < 0.001 vs. C3a (−) & SB203580 (−) group; # p < 0.05, ## p < 0.01 vs. C3a ( +) & SB203580 (−) counterparts; ns: not significant. Images are presented, and values are expressed as the mean ± SEM from three independent experiments

Article Snippet: For detecting C3, anti-human/mouse C3 polyclonal antibody (GeneTex) was used, and for detecting C3a, the neoepitope recognizing antibodies were used as follows: anti-human C3a monoclonal antibody, clone 2991 (Bio-Rad) and anti-mouse C3a monoclonal antibody, clone I87-1162 (BD Biosciences). a Representative IHC staining of intact C3 (left) and C3a (right) in human Hh-type MB tissue; brown color displays the positive staining.

Techniques: Cell Culture, In Vitro, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Complement C3a activates astrocytes to promote medulloblastoma progression through TNF-α

doi: 10.1186/s12974-022-02516-9

Figure Lengend Snippet: C3a-activated astrocytes promote MB cell proliferation through TNF-α secretion. a , b Purified MB cells were cultured in vitro in the presence of recombinant TNF-α, IL-6, or vehicle control (Ctrl) for 48 h and then immunostained for Ki67 (red). DAPI was used to counterstain the cell nuclei ( a ). The percentage of Ki67 + cells was quantified ( b ). *** p < 0.001, ns: not significant vs. Ctrl group. c , d Astrocytes were pretreated with 100 nM C3a or vehicle control for 24 h, washed thoroughly, and cultured for another 24 h for CM collection. Then, purified MB cells were cultured with the CM above together with the TNF-α receptor antagonist R-7050 (500 nM) or not for 48 h. Ki67 was immunostained ( c ), and the percentage of Ki67 + cells was quantified ( d ). *** p < 0.001, ns: not significant vs. Ctrl CM group, # p < 0.05 vs. (C3a-AS CM + vehicle) group. Images are presented, and values are expressed as the mean ± SEM from three independent experiments

Article Snippet: For detecting C3, anti-human/mouse C3 polyclonal antibody (GeneTex) was used, and for detecting C3a, the neoepitope recognizing antibodies were used as follows: anti-human C3a monoclonal antibody, clone 2991 (Bio-Rad) and anti-mouse C3a monoclonal antibody, clone I87-1162 (BD Biosciences). a Representative IHC staining of intact C3 (left) and C3a (right) in human Hh-type MB tissue; brown color displays the positive staining.

Techniques: Purification, Cell Culture, In Vitro, Recombinant, Control

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3a and C5a aggravate inflammation in HRPE cells via the NF-κB signaling pathway. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) or C5a (1 µg/ml) with or without NF-κB inhibitor PDTC (10 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was measured by ELISA. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. PGE2, prostaglandin E2; HRPE, human retinal pigment epithelium.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3aR and C5aR antagonist inhibit inflammation and NF-κB signaling in HRPE cells challenged with complement C3a and C5a. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was determined by ELISA. HRPE cells were treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM), and the release of (F) TNF-α, (G) IL-1β, (H) IL-6, (I) PGE2 and (J) IL-10 was determined by ELISA. (K) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM) were determined by western blot. (L) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM) were determined by western blot. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. p-NF-κB, phosphorylated NF-κB; C5aR, C5a receptor; HRPE, human retinal pigment epithelium; CCX, CCX168; SB, SB290157.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Expressing, Western Blot, Control

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 2: Te concentrations of complement pyrolysis products were found higher in pleural efusion than in plasma from TPE patients. (a) Complement pyrolysis products, including C3a, C3b, C3d, C5a, and opsonin receptors (CR1 and CR3) were detected in human tuberculosis pleural biopsy samples by immunohistochemistry. (Original magnifcation, ×200) (n 4). (b) Higher levels of complement pyrolysis products were found in pleural efusion than in plasma in TPE patients (n 20). Te concentrations of complement pyrolysis products in pleural fuid and plasma from TPE patients were measured by ELISA (n 20).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Clinical Proteomics, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 3: Monocyte migration was inhibited by antibodies that blocked CXCL12. (a) CXCL12 and CXCR4 staining by immunohisto- chemistry in human pleural biopsy (original magnifcation, ×200) (n 4). (b) Te concentration of CXCL12 in pleural fuid and plasma from TPE patients was measured by ELISA (n 20). (c) CXCL12 produced by PMCs was measured by PCR and ELISA after Mpt64 and anaphylatoxin activation. PMCs were incubated for 24 hours in control media or in media with Mpt64 (20 μg/ml) and with or without human C3a (100 nM) or C3aRA (100 nM) (n 4). (d) Coexpression of CXCL12-CXCR4 in PMCs and monocytes from TPE was detected by immunofuorescence (original magnifcation, ×400) (n 4). (e) Monocytes were seeded into the top chamber of a transwell system, and the supernatant from PMCs cultured with anti-CXCL12 antibody or PBS were placed in the bottom chamber. Te migratory index was calculated by dividing the number of monocytes that migrated in response to the supernatants from cultured PMCs by the number of monocytes that migrated in response to the control. ∗vs the MO-PBS group, ∗∗P < 0.01. #vs the MO-PMC group, ##P < 0.01 (n 4).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Migration, Staining, Immunohistochemistry, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Produced, Activation Assay, Incubation, Control, Cell Culture

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) IFN-γ secretion (left) and intracellular C3a generation (right) by healthy donor CD4+ T cells activated as shown in the presence and absence of ICAM-1 with and without a cell-permeable cathepsin L inhibitor (CTSLi) for 72h (n=4 independent experiments). (B) Representative flow cytometry plots from n=4 independent experiments showing C3 mRNA and the mTOR down-stream target S6 kinase phosphorylated at ser235/ser236 (p-S6) in healthy donor CD4+ T cells activated as shown in the presence or absence of ICAM-1 for 36h. (C) Extracellular acidification rate (ECAR, a measure of glycolysis) and oxygen consumption rate (OCR, a measure of oxidative phosphorylation) in healthy donor naive CD4+ T cells activated as shown in the presence or absence of ICAM-1 with and without a CTSL inhibitor for 36h. Shown are representative ECAR (above) and OCR (middle) graphs together with cumulative data of the maximal respiration and glycolysis (below) from n=3 independent experiments. NA, non-activated; FCCP, p-trifluoromethoxyphenyl hydrazone; OM, oligomycin; ROT, rotenone. Bars show mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. See also Figure S4.

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Flow Cytometry, Phospho-proteomics

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: (A) Steady-state expression of C3 and IFNG mRNA in paired CD4+ T cells drawn from blood and synovial fluid of pediatric patients with juvenile idiopathic oligo-arthritis (n=4; Table S2). Bars show mean + SEM. (B) IFN-γ secretion by paired CD4+ T cells drawn from blood and synovial fluid of two patients with rheumatoid arthritis (RA) activated in vitro with antibodies against CD3 with, or without, ICAM-1. (C-D) C3 mRNA expression by synovial T cells of patients with either oligo-arthritis (OA), non-inflamed RA or inflamed RA derived from an independent dataset (Zhang et al., 2019) (C) and receiver operating characteristic curves showing performance of C3 and IFNG mRNA expression in synovial T cells as biomarkers to distinguish inflamed from un-inflamed RA (D). (E) LFA-1 expression (top), C3 mRNA (middle) and IFN-γ secretion (bottom) by peripheral blood CD4+ T cells from patients with LFA-1 mutations (LAD-1 disease) (see Table S3) and age- and sex-matched controls activated in vitro as shown with, and without ICAM-1 for 72h. Data are from three patients, two of whom donated twice, and five controls. Bars represent mean of duplicate measurements per subject. (F) regression lines showing correlation between IFN-γ and LFA-1 expression (left) and between C3 mRNA and LFA-1 expression (right) from primary data in (E). 95% confidence intervals of the regression line are shown and individual donors are marked. (G) IFN-γ secretion by peripheral blood CD4+ T cells from three patients with LAD-1 disease after transduction with control adenovirus or adenovirus encoding C3a, electroporation with mRNA encoding GFP or C3a or electroporation with BSA or C3H2O protein, as indicated. Cells were activated with anti-CD3+CD46+ICAM-1 after transduction or electroporation. Each patient has been labelled, two of whom donated three times, and the over-expression method shown by color-coding. *p<0.05. See also Figure S6 and Tables S2 and S3.

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Expressing, In Vitro, Derivative Assay, Transduction, Control, Electroporation, Over Expression

Journal: Immunity

Article Title: Diapedesis-induced integrin signalling via LFA-1 facilitates tissue immunity by inducing intrinsic complement C3 expression in immune cells

doi: 10.1016/j.immuni.2020.02.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified human C3a , CompTech , Cat. #A118.

Techniques: Virus, Generated, Control, Isolation, Recombinant, Purification, Staining, Diagnostic Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Labeling, Gene Expression, Reverse Transcription, Synthesized, Software, Transfection, Imaging, Flow Cytometry, Microscopy, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Receptors for C3a and C5a modulate stability of alloantigen-reactive induced regulatory T cells 1

doi: 10.4049/jimmunol.1300847

Figure Lengend Snippet: (A) Representative flow plots (left and middle, 12,000 events shown in each plot) and total number (right) of Foxp3+ cells generated from CFSE-labeled CD4+ WT or C3ar1−/−C5ar1−/− Tconv. (B) Suppression capacities of WT (solid line) or C3ar1−/−C5ar1−/− (dashed line) allo-iTreg. (C) Total Foxp3GFP+ cells generated as in (A) ± blocking anti-C3a/C5a mAb. (D) Percentages (left/middle) and total numbers (right) of Foxp3+ cells generated as in (A) using allogeneic WT or Daf1−/− DCs. (E) Splenic frequencies of Foxp3GFP+ (left) and total CD4+ cells (right) 14d after adoptive transfer of naive WT or C3ar1−/−C5ar1−/− CD4+ cells into syngeneic rag1−/− mice. (F) Immunoblot for pFoxo1 and total Foxo1 from stimulated iTreg cell lysates. Results are each representative of 3 independent experiments. Error bars indicate mean ± s.d. *p<0.05

Article Snippet: We purchased recombinant human C3a and C5a from R&D Systems (Minneapolis, MN USA), rat IgG2bκ and blocking anti-C3a and anti-C5a mAb from BD Biosciences (Franklin Lakes, NJ USA), C3aR antagonist SB290157 and AKT inhibitor VIII from Calbiochem (San Diego, CA USA), and C5aR peptide antagonist [Ac-Phe-cyclo(Orn-Pro-dCha-Trp-Arg)] from GenScript (Piscataway, NJ USA).

Techniques: Generated, Labeling, Blocking Assay, Adoptive Transfer Assay, Western Blot