Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Expression of Gα subunits in macrophages and roles of Gnai2 and Gnai3 in complement C5a-mediated chemotaxis. A, expression levels of Gα subunits in mouse resident peritoneal F4/80+ cells (macrophages). RNA-Seq analysis was performed using RNA isolated from resident peritoneal F4/80+ cells purified by cell sorting (n = 3 mice). Inset (superimposed graph with an interrupted y axis), expression levels of receptors for complement components 3a and 5a. Error bars, S.E. B, schematic diagram showing C5aR, a member of the G protein–coupled receptor superfamily, and a heterotrimeric G protein in which the subunits are color-coded blue (Gα), green (Gβ), and white (Gγ). The four Gα families (Gαi/o, Gαs, Gαq/11, and Gα12/13) are listed below the blue α-subunit (Gα) together with the names of the corresponding genes investigated with knockout mouse models, including two genes encoding β-subunits: Gnb1 (Gβ1) and Gnb2 (Gβ2). C, migration plots of WT, PTX-treated WT, Gnai2−/−, and Gnai3−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. The chemotaxis index is also known as the y-forward migration index and has a range of −1 to +1. *, p < 0.05; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 75 for each group, except n = 50 for the WT + PTX group; three independent experiments, except two independent experiments for the WT + PTX group).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Expressing, Chemotaxis Assay, RNA Sequencing, Isolation, Purification, FACS, Knock-Out, Migration, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-induced Ca2+ transients and lamellipodial membrane protrusions are not impaired in Gnai2−/− or Gnai3−/− macrophages. A, simultaneous imaging of intracellular [Ca2+] (green trace) and projected cell area (black trace) in individual WT and Gnai2−/− macrophages challenged with 20 nm complement C5a. Intracellular [Ca2+] is indexed as relative Cal-510 fluorescence intensity (F/F0) in which the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, summary box plots of peak complement C5a-induced Ca2+ transients and projected cell area. *, p < 0.05; n.s., not significant; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 50 (WT), n = 19 (WT + PTX), n = 46 (Gnai2−/−), and n = 9 (Gnai3−/−); 2–3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, Imaging, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Sequestration of intracellular [Ca2+] with EGTA does not prevent complement C5a-induced lamellipodial membrane protrusions. A, example (green trace) of a complement C5a-induced Ca2+ transient largely blocked in a WT macrophage after passively loading the cell with the Ca2+ chelator EGTA using its AM ester form (EGTA/AM). The box plots on the right show peak complement C5a-induced Ca2+ transients measured in the absence and presence of EGTA/AM. B, the trace shows the projected cell area corresponding to the above Ca2+ trace (A). The box plots on the right show peak complement C5a-induced cell spreading in the absence and presence of EGTA/AM. *, p < 0.05; n.s., not significant; Mann–Whitney U test (n = 50 (WT pool) and n = 43 (WT + EGTA/AM); n = 3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-mediated chemotaxis is preserved in Gnaq/Gna11 double knockout and Gna12/Gna13 double knockout macrophages. A, schematic diagram highlighting genes of the Gαq/Gα11 (Gnaq and Gna11) and Gα12/Gα13 (Gna12 and Gna13) families of Gα subunits that potentially may be activated by C5aR. B, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index). *p < 0.05; Kruskal–Wallis test and post-hoc Mann–Whitney U test with Bonferroni correction (n = 75 for each group; 3 independent experiments). C, migration plots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. D, 200 × 300-μm snapshots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. Black arrows, elongated trailing ends. The schematic diagram on the left shows a μ-Slide Chemotaxis chamber with one of the two 40-μl reservoirs (filled with a blue dotted pattern) containing 20 nm complement C5a. E, box plots of maximal tail lengths developed by macrophages migrating in a chemotactic complement C5a gradient over a 6-h period. *, p < 0.05; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 50 cells/group; sampled from two independent experiments). F, representative example, from two independent experiments, of RhoA activity measured using a colorimetric G-LISA assay, in which active RhoA (RhoA-GTP) was indexed as absorbance at 490 nm (A490). RhoA protein was used as positive control. Bars, mean ± S.D. (error bars) of duplicate measurements.

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Chemotaxis Assay, Double Knockout, MANN-WHITNEY, Migration, Activity Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: UTP- and complement C5a-induced Ca2+ transients in Gnaq/Gna11 double knockout macrophages. A, time-lapse images (90 × 90 μm) of WT macrophages loaded with the fluorescent Ca2+ indicator Cal-520. UTP was added as indicated. Scale bar, 10 μm. The intracellular Ca2+ signaling corresponding to the labeled macrophage (MΦ1) is shown below. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, time-lapse images (90 × 90 μm) of Gnaq/Gna11 dKO macrophages loaded with the fluorescent Ca2+ indicator Cal-520. UTP was added as indicated, and 22 min later, complement C5a was applied to the same cells. Scale bars, 10 μm. The intracellular Ca2+ signals corresponding to the labeled macrophages (MΦ1, MΦ2, and MΦ3) are shown below.

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Double Knockout, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Intact complement C5a-induced Ca2+ transients in Gna12/Gna13 double knockout and Gnaq/Gna11 double knockout macrophages. A, time-lapse images (90 μm × 90 μm) of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. Scale bars, 10 μm. B, intracellular Ca2+ signals corresponding to the above labeled macrophages (MΦs; A). Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. C, summary peak [Ca2+] data. n.s., not significant; Kruskal–Wallis test (n = 20–27 per group; 2–3 independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Double Knockout, Labeling, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gna15 is redundant for complement C5a-mediated chemotaxis. A, schematic diagram highlighting that Gna15 belongs to the Gαq/Gα11 family of α-subunits. B, migration plots of WT and Gna15−/− macrophages in a chemotactic complement C5a gradient. C, 200 × 300-μm snapshot of WT and Gna15−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. n.s., not significant; Mann–Whitney U test (n = 75 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Chemotaxis Assay, Migration, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-induced Ca2+ transients are largely abolished in Gna15-deficient macrophages. A, time-lapse images (90 × 90 μm) of WT macrophages loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a and UTP were added as indicated. Scale bar, 10 μm. Below the series of four images is the intracellular Ca2+ signaling corresponding to the macrophage (MΦ) labeled MΦ1. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, time-lapse images (90 μm × 90 μm) of Gna15−/− macrophages loaded with Cal-520. Complement C5a and UTP were added as indicated. Scale bar, 10 μm. Below are traces corresponding to the labeled Gna15−/− macrophages (MΦ1 and MΦ2, respectively). C, summary peak [Ca2+] data. *, p < 0.05; Mann–Whitney U test (n = 14 for WT (two independent experiments); n = 30 for Gna15−/− (three independent experiments)).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Intact complement C5a-induced lamellipodial membrane spreading and Ca2+ transients in Gna15−/− macrophages. A, time-lapse images (90 × 90 μm) of WT and Gna15−/− macrophages stained with the fluorescent plasma membrane marker CellMask Orange and loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. The white arrows indicate examples of lamellipodial membrane protrusion. Scale bars, 10 μm. Below is the intracellular Ca2+ signaling corresponding to the Gna15−/− macrophage labeled MΦ1. Complement C5a and UTP were added as indicated. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, box plots of projected cell area before and after application of complement C5a to WT or Gna15−/− macrophages. *, p < 0.05; Mann–Whitney U test. C, box plots of relative peak projected cell area after application of ligand-free medium (Sham) and complement C5a-containing medium to WT and Gna15−/− macrophages. *, p < 0.05; Mann–Whitney U test (n = 34 for WT and n = 29 for Gna15−/− (three independent experiments)). D, box plots of the changes in cell area (prestimulation cell area subtracted from the peak poststimulation cell area) after stimulating WT and Gna15−/− macrophages with complement C5a (n.s., not significant; Mann–Whitney U test).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Membrane, Staining, Clinical Proteomics, Marker, Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gnb2−/− macrophages show robust complement C5a-induced cell spreading and Ca2+ transients. A, time-lapse images (90 × 90 μm) of WT and Gnb2−/− macrophages stained with the fluorescent plasma membrane marker CellMask Orange and loaded with the fluorescent Ca2+ indicator Cal-520. Complement C5a was added as indicated. The white arrows indicate examples of lamellipodial membrane protrusion. Scale bars, 10 μm. Below the series of cell morphology (CellMask Orange) images is the intracellular Ca2+ signaling corresponding to the individual macrophages labeled MΦ1. Complement C5a and UTP were added as indicated. Intracellular [Ca2+] is indexed as relative Cal-520 fluorescence intensity (F/F0), where the measured fluorescence intensity (F) is divided by the resting fluorescence intensity (F0) after subtracting the background fluorescence intensity at each time point. B, summary box plots of peak cell spreading and peak intracellular [Ca2+] induced by complement C5a. n.s., not significant; Mann–Whitney U test (n = 52 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Staining, Clinical Proteomics, Membrane, Marker, Labeling, Fluorescence, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Gnb2−/− macrophages have decreased velocity and impaired navigation in a chemotactic complement C5a gradient. A, schematic diagram highlighting the Gβ-subunit Gβ2 (Gnb2). B, migration plots of WT and Gnb2−/− macrophages in a chemotactic complement C5a gradient. C, 200 × 300-μm snapshot of WT and Gnb2−/− macrophages in a chemotactic complement C5a gradient. D, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index), calculated by dividing the displacement along the y axis by the cumulative distance migrated. *, p < 0.05; Mann–Whitney U test (n = 75 per group; three independent experiments).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Migration, Chemotaxis Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Tabular summary and schematic diagram of G protein subunits involved in transducing complement C5a gradients into directed migration. A, tabular summary of results. B, schematic summary. C5aRs couple (i) directly to at least two heterotrimeric G proteins formed by Gα15 and Gαi2 subunits and possibly also Gα12/Gα13 and Gαi3 (not shown) subunits and their respective Gβγ subunits and (ii) indirectly to Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP signaling, which stimulates P2Y2Rs. The Gαi2 subunit is indispensable for chemotaxis and associates with Gβ2-containing, or possibly also Gβ1-containing, Gβγ subunits. Gαi2/Gβ2γx heterotrimeric G proteins, where x is unknown, dissociate into active (GTP-bound) Gαi2 subunits and Gβ2γx dimers following receptor activation by complement C5a. The Gβ2γx (or possibly Gβ1γx) dimers activate PI3Ks, which catalyze the conversion of PIP2 to PIP3. PIP3 is known to recruit pleckstrin homology domain–containing Rac- and Cdc42-GEFs to the membrane. Activation of Gα15-containing heterotrimeric G proteins directly by complement C5a, as well as indirect activation of Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP and UTP signaling, increases the activity of PLC-β isoforms, which catalyze the hydrolysis of PIP2 to inositol IP3 and diacylglycerol. IP3 induces Ca2+ release from the endoplasmic reticulum, but this Ca2+ signal is largely redundant for lamellipodial membrane protrusions and chemotaxis. However, we speculate that depletion of PIP2 by PLC-β isoforms and PI3Ks contributes to the formation of lamellipodial membrane protrusions by promoting the dissociation of Rac– and Cdc42-GTPase–activating proteins (GAPs). We speculate that activation of Gα12/Gα13 by complement C5a-C5aR signaling, which remains to be confirmed, increases the activity of the monomeric (small) G proteins RhoA and RhoB via RhoGEFs. Activated (GTP-bound) RhoA and RhoB promote actomyosin-dependent retraction of the trailing end of migrating cells, whereas the RhoGAP Myo9b is thought to inhibit RhoA and RhoB at the front of cells. Extracellular ATP and UTP stimulate P2Y2Rs. ATP, but not UTP, additionally activates P2X receptors (not shown), ligand-gated cation channels. ATP and UTP are rapidly degraded by surface ectonucleotidases, such as CD39, to form ligands for other purinergic receptors (not shown).

Article Snippet: The final concentration of complement C5a was 20 n m . Complement C5a (2150-C5-025, R&D Systems, Abingdon, UK) was reconstituted in PBS containing 0.1% BSA, which had been filtered using a 0.2-μm cellulose acetate membrane (723-2520, Thermo Fisher Scientific).

Techniques: Migration, Chemotaxis Assay, Activation Assay, Membrane, Activity Assay

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Morphological appearance of DPSCs cultured with different concentration of C5a for 2 weeks. A 50, B 100, C 200, D 300, E 400 ng/ml C5a groups and F Control group. DPSCs, dental pulp mesenchymal stem cells; C5a, complement component 5a

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Concentration Assay, Control

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Effect of different concentration of C5a stimulation on DSPP protein expression in DPSC. All 6 groups were cultured in dentinogenic medium. DSPP expression was determined by Western blot after 28 days of culture time. Results are expressed as relative expression to Actin. Data are presented as mean ± SEM of 3 independent experiments. *P < 0.05, *showed 100 ng/ml and 200 ng/ml groups expressed significantly lower DSP protein compared with other groups.. △ P < 0.05, △showed 400 ng/ml group expressed significantly higher DSP protein. DSPP, dentin sialophosphoprotein; DPSC, dental pulp mesenchymal stem cell

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Concentration Assay, Expressing, Cell Culture, Western Blot

Journal: BMC Oral Health

Article Title: Different concentrations of C5a affect human dental pulp mesenchymal stem cells differentiation

doi: 10.1186/s12903-021-01833-4

Figure Lengend Snippet: Cell morphology and mineralized nodules of hDPSCs cultured with C5a and mineralized medium for 28 days. A 50 ng/ml C5a culture hDPSCs (arrows nodules). B 100 ng/ml C5a culture hDPSCs displayed smaller and less mineralized nodules (arrows nodules). C 200 ng/ml C5a culture hDPSCs, displayed smaller and less mineralized nodules (arrows nodules). D 300 ng/ml C5a culture hDPSCs (arrows nodules). E 400 ng/ml C5a culture hDPSCs displayed bigger and more mineralized nodules (arrows nodules). F Control group. C5a, complement component 5a, hDPSCs, human dental pulp mesenchymal stem cell (arrows nodules). G Quantification of mineralized nodule formation. Data are shown as mean OD/µg of total protein ± SD (n = 6). *Indicates significant differences compared to conrol group (P < 0.05)

Article Snippet: Cells were cultured in a 37 °C, 5% CO 2 incubator and divided into six groups (n = 1 × 10 5 cells/group, using Corning® 25cm 2 Rectangular Canted Neck Cell Culture Flask with Vent Cap): (i) cells treated with basic cell culture medium containing 10 nmol/l dexamethasone, 5 mmol/l β-glycerophosphate and 50 mg/ml vitamin-C-phosphate as control; (ii) DPSCs treated with 50 ng/ml recombinant human complement component C5a protein (C5a; R&D Systems, Inc.); (iii) DPSCs treated with 100 ng/ml C5a; (iv) DPSCs treated with 200 ng/ml C5a; (v) DPSCs treated with 300 ng/ml C5a; and (vi) DPSCs treated with 400 ng/ml C5a.

Techniques: Cell Culture, Control

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

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Article Snippet: This was followed by 4 washes and incubation with 100 ng of the detection antibody, biotinylated mouse monoclonal anti-human C5a (clone 295009, R&D Systems) for 60 minutes at room temperature with shaking.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

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

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Article Snippet: This was followed by 4 washes and incubation with 100 ng of the detection antibody, biotinylated mouse monoclonal anti-human C5a (clone 295009, R&D Systems) for 60 minutes at room temperature with shaking.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: FGF19-Induced Inflammatory CAF Promoted Neutrophil Extracellular Trap Formation in the Liver Metastasis of Colorectal Cancer.

doi: 10.1002/advs.202302613

Figure Lengend Snippet: Figure 5. FGF19 induced NET formation by facilitating complement C5a and IL-1𝛽production in iCAFs. A) Cytokine array of the media of LX-2 cells pretreated with rhFGF19 (50 ng mL−1) for 24 h. Seven factors were upregulated in the supernatant of LX-2 cells stimulated with rhFGF19. B) Quantification of NETs formed by neutrophils stimulated with complement C5a, CXCL11, IL-1𝛼, IL-1𝛽, IL-18, MIF, or PAI-1 (n = 18 RMFs from 6 experimental replicates per group). C) Intracellular and D) extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with rhFGF19 or FGF19-containing CM (n = 3 for WB, or 6 for ELISA). E) Quantification of NETs formed by neutrophils cocultured with LX-2 cells that had been pretreated with FGF19-containing CM in the presence of C5a neutralizing antibody (50 ng mL−1) or IL-1𝛽neutralizing antibody (1 μg mL−1) (n = 18 RMFs from 6 experimental replicates per group). F) Representative IHC staining of C5a or IL-1𝛽expression in paired primary CRC and CRLM tissues. Scale bar: 50 μm. G) Expression of complement C5a and IL-1𝛽were higher in CRLM tissues (n = 30) than that in paired primary CRC tissues (n = 30). H) Correlation between IHC scores of FGF19 and C5a or IL-1𝛽in human CRLM tissues (n = 30). I) ChIP‒qPCR assays showed the recruitment of STAT3 to the promoter regions of C5 and IL1B (n = 3). J,K) Intracellular and L) extracellular expression of C5a and IL-1𝛽in LX-2 cells stimulated with rhFGF19 or FGF19-containing CM and treated with or without fisogatinib (100 nм), fedratinib (10 μм), C188-9 (5 μg mL−1), and anakinra (20 mg mL−1) (n = 3 for WB, or 6 for ELISA). M) Extracellular expression of complement C5a and IL-1𝛽in LX-2 cells treated with FGF19-containing CM for 24 h and then subjected to regular medium, regular medium with rhIL-1𝛼(1 ng mL−1), FGF19-free CM, fresh FGF19-containing CM, or anakinra for 24 h (n = 6). ** or ##p < 0.01; *** or ###p < 0.001; n.s., nonsignificant. In (B), (D), (I), (L), and (M), data were subjected to Student’s t-test. In (E), data were subjected to Mann–Whitney test. In (G), data were subjected to paired Student’s t-test. See also related Figure S6, Supporting Information.

Article Snippet: Human FGF19 neutralizing antibody (AF969, R&D Systems), human IL-1β neutralizing antibody (AF-201-NA, R&D Systems), and human complement C5a neutralizing antibody (MAB2037, R&D Systems) were used in this study.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Inflammation

Article Title: Red Blood Cell-Derived Exosomes Deliver Complement C5 to Exacerbate Neuroinflammation and Neuronal Injury after Intracerebral Hemorrhage

doi: 10.1007/s10753-026-02456-z

Figure Lengend Snippet: ICH-Exos C5 promoted necroptosis and M1 polarization in hemin-induced microglia. ( A ) The heat map shows the differentially expressed proteins. ( B ) C5a level in brain tissue analyzed by IHC. * p < 0.05 vs. sham group. ( C ) C5a level in serum was detected by ELISA. * p < 0.05 vs. sham group. Under RIPA lysis conditions, the results of Western blot for C5a in ( D ) human RBC-Exos and ( E ) murine RBC-Exos after untreated EVs or after treatment with proteinase K (5 U/mL, 10 min) . # p < 0.05 vs. Sham-Exos/Normal-Exos group; @ p < 0.05 vs. ICH-Exos group. ( F ) C5a level in HMC3 cells was analyzed by Western blot and ELISA. ( G ) Cell viability was detected by CCK-8. ( H ) Cell apoptosis was detected by flow cytometry. ( I ) p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, and MLKL levels were detected by Western blot. ( G ) The proportions of CD86 + M1 and CD163 + M2 macrophages were detected by flow cytometry. ( K ) iNOS, COX-2, CCL2, ARG1, CD206, and YM1 levels analyzed by qRT-PCR. ( L ) iNOS, COX-2, ARG1 and CD206 levels analyzed by Western blot. (M) ELISA was used to detect IL-1β, IL-6, and TNF-α levels. n = 3. # p < 0.05 vs. Normal-Exos group; & p < 0.05 vs. ICH-Exos group

Article Snippet: The mouse TNF-α (CSB-E04741m, CUSABIO, China), human TNF-α (KE00154, Proteintech), IL-1β (KE10003, KE00021, Proteintech), IL-6 (CSB-E04639m, CSB-E04638h, CUSABIO), and C5a (CSB-E08512h, CUSABIO) ELISA kits were operated according to the manufacturer’s instructions.

Techniques: Paraffin-embedded Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Lysis, Western Blot, CCK-8 Assay, Flow Cytometry, Quantitative RT-PCR

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Joint IM induces destruction of the synovialis and cartilage in rats. Hematoxylin and eosin staining was performed. Magnification, ×200. IM, immobilization; C5a, complement component 5a.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: Staining

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Effects of joint IM on collagen accumulation in the synovialis and cartilage. Masson staining was performed. Magnification, ×200. IM, immobilization; C5a, complement component 5a.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: Staining

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Apoptosis of synovial and cartilage cells is observed in rats with joint IM. Terminal deoxynucleotidyl transferase dUTP nick end labeling was performed. Magnification, ×200. IM, immobilization; C5a, complement component 5a.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: TUNEL Assay

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Effects of joint IM on serum C5a and pro-inflammatory cytokine expression levels. (A) Expression levels of C5a, IL-1β, TNF-α and IL-17A in the serum; (B) expression levels of C5a, IL-1β, TNF-α and IL-17A in joint cavity fluid. The graphs present the mean ± SEM. *P<0.05, **P<0.01 vs. control group at the same time point. The data are presented as the average of triplicate values. Error bars represent the SEM. IM, immobilization; C5a, complement component 5a; SEM, standard error of the mean; IL, interleukin; TNF-α, tumor necrosis factor-α.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: Expressing, Control

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Joint IM increases the expression of C5a in the synovialis and cartilage. Immunohistochemical staining of C5a and Masson staining was performed. Magnification, ×200. IM, immobilization; C5a, complement component 5a.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Molecular Medicine Reports

Article Title: C5a aggravates dysfunction of the articular cartilage and synovial fluid in rats with knee joint immobilization

doi: 10.3892/mmr.2018.9208

Figure Lengend Snippet: Expression levels of C5a in the synovialis and cartilage of rats with knee joint IM at different time points. (A) C5a mRNA expression; (B) Representative bands of C5a protein in the synovialis of rats at different time points; (C) Representative bands of C5a protein expression in the cartilage at different time points. The graphs illustrate the mean ± SEM. *P<0.05, **P<0.01 vs. control group at the same time point. The data are presented as the average of triplicate values. Error bars represent the SEM. IM, immobilization; C5a, complement component 5a; SEM, standard error of the mean; IL, interleukin; TNF-α, tumor necrosis factor-α.

Article Snippet: The Rat IL-1β ELISA kit (cat. no. E-EL-R0012c), Rat C5a ELISA kit (cat. no. E-EL-R0257c), Rat TNF-α ELISA kit (cat. no. E-EL-R0019c) and Rat IL-17A ELISA Kit (cat. no. E-EL-R0566c; all Elabscience, Houston, TX, USA) were used.

Techniques: Expressing, Control