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: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial adhesion to the RTEC. RTEC were pre-treated with C5a (as indicated or otherwise 10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to assessment of bacteria binding to RTEC. (A,B) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (C,D) Bacterial adhesion to RTEC, evaluated by fluorescence microscopy analysis. (C) Representative fluorescence images of tri-iodothyronine and tetramethylrhodamine-labeled J96 adhesion to RTEC, J96 (red), and 4’,6-diamidino-2-phenylindole (blue). Scale bars, 50 µm. (D) Quantification of bacteria adhesion to RTEC corresponding to the images in (C) . Results were expressed as number of bacteria per 10 3 RTEC. (A,B,D) data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test [ (A) n = 12 individual wells per group, (B) n = 8 individual wells per group, (C) n = 6 individual images (×200 magnification) from two coverslips per group]. All results shown are representative of three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial internalization into the RTEC. (A) Bacterial uptake by RTEC, evaluated by bacterial plate count assay. RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to the assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test ( n = 8 individual wells) and representative of three independent experiments. (B,C) Bacterial uptake by RTEC, evaluated by fluorescence microscopy analysis. RTEC were pre-treated with or without C5a (10 nM) for 24 h and subjected to the assay. (B) Representative fluorescence images of (tri-iodothyronine and tetramethylrhodamine-labeled) bacteria internalization into RTEC. Bacteria (red), F-actin (green), and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. Lower panel image corresponding to the boxed region in the top-right image shows the cross-sectional views in z stack (bottom and side panel) of RTEC, F-actin, and bacteria, demonstrating J96 within the cytoplasm of RTEC. A representative of three experiments is shown. (C) Quantification of bacteria uptake by RTEC corresponding to the images in (B) . Results were expressed as number of bacteria per 10 3 RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 15 individual images per group). * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Fluorescence, Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial transmigration through the monolayer of RTEC. Bacterial transmigration in RTEC that had been pre-treated with or without C5a (10 nM) for 24 h, then incubated with or without bacteria up to 3 h. (A) Transmigrated bacteria recovered from the lower chamber of RTEC cultures, evaluated by plate count assay. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 4 individual wells per group). (B–D) Epithelial barrier damage, evaluated at 2 h after incubation with bacteria by reduction of tight junction marker ZO-1 expression. (B) Western blot analysis for ZO-1 in RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 3 individual images per group). (C) Representative fluorescence images of ZO-1 (green) staining in RTEC. Scale bars, 10 µm. (D) Quantification of ZO-1-positive cells, shown as C5a treatment relative to control, corresponding to the RTEC shown in (C) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 4 individual images (×200 magnification) per group]. * P < 0.05, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Transmigration Assay, Incubation, Bacteria, Marker, Expressing, Western Blot, Two Tailed Test, Fluorescence, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: C5a stimulation upregulates Man expression in renal tubular epithelial cell (RTEC). RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) or with C5a, in the presence of C5aR1 antagonist (PMX53, 5 µM) or vehicle (Ctrl) for 24 h and subjected to assessment of Man expression and bacteria binding to RTEC. (A,B) Fluorescence microscopy analysis. (A) Representative fluorescence images of Man expression in RTEC (non-permeabilized). Man (green) detected by fluorescein-labeled Galanthus nivalis lectin (GNL) and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (B) Quantification of Man expression, shown as relative fluorescence intensity in GNL-positively stained area corresponding to the images shown in (A) . (C,D) Fluorescence intensity-based microplate assay. (C) Man expression in RTEC that had been treated with C5a and/or LPS. (D) Man expression in RTEC that had been treated with C5a and PMX53. (E) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (B–D) Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [ (B) n = 6 coverslips per group, under ×100 magnification, (C) n = 9 individual wells per group, (D,E) n = 6–8 individual wells per group]. All results shown are representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: Ctrl, control; RFI, relative fluorescence intensity; RFU, relative fluorescence units.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Expressing, Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Uropathogenic Escherichia coli adhesion to renal tubular epithelial cell (RTEC) through binding to Man on the cell surface of the RTEC. (A) Confocal microscopic images of bound bacteria in RTEC (non-permeabilized) that had been incubated with labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin and 4’,6-diamidino-2-phenylindole (blue) are shown. Left image: compressed image. Scale bars, 10 µm. Right image: corresponding to the boxed region in the left image show the cross-sectional views in z stack (bottom and side panel) of RTEC, Man, and bacteria, demonstrating association of Man and J96 at cell surface of RTEC. (B) Bacteria binding to RTEC, evacuated by bacterial plate count assay. RTEC were incubated with d -mannose or glucose (1 or 5%) for 30 min before the addition of bacteria. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 8 individual wells per group). (C,E) Man expression in RTCE that had been pre-treated without C5a (C) or with C5a (E) for 24 h and then treated with buffer alone (PBS) or containing α-mannosidase (5 mM) or control enzyme (β-galactosidase) for 1 h, evacuated by fluorescence intensity-based microplate assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 6 individual wells per group). (D,F) Bacterial binding to RTEC that had undergone the same treatments as described in (C,E) , evacuated by bacterial plate count assay. (D) Without C5a pre-treatment. (F) With C5a pre-treatment. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 8 individual wells per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Binding Assay, Bacteria, Incubation, Labeling, Expressing, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Intracellular signaling/molecules responsible for the action of C5a on Mannosyl residue expression. (A,B) Western blot analysis for ERK1/2 and IκB phosphorylation in renal tubular epithelial cell (RTEC) after C5a (10 nM) stimulation for up to 60 min. In each set of blots, the top row of bands corresponds to incubating membrane with appropriate anti-phospho-antibody and the bottom row of bands corresponds to incubating membrane with appropriate total antibody. Relative amounts of protein phosphorylation are shown in the lower panel of each set of blots. Data were analyzed by one-way ANOVA ( n = 3/group, resulting from three independent experiments). (C) Effect of inhibition of ERK1/2 pathway on Man expression in RTECs assessed by fluorescence intensity-based microplate assay. RTEC monolayers were pre-incubated with C5a for 24 h in the presence of ERK1/2 pathway inhibitor (U0126, 10 μM) and the vehicle control (DMSO) then were used for quantification of Man expression. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9–12 individual wells per group). A representative result of three experiments is shown. (D,E) Production of TNF-α in RTEC that had been treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to RT-qPCR (D) and ELISA (E) . Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 3/group, resulting from three independent experiments). (F) Man expression in RTEC that had been pre-treated with recombinant human TNF-α (10 ng/mL) or vehicle control (BSA) for 24 h, evaluated by fluorescence intensity-based microplate assay. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9 replicate wells/group). A representative result of three experiments is shown. (G,H) Bacterial adhesion to RTEC. (G) Representative microscopic images of bacteria adhesion to RTEC that had been pre-treated with TNF-α for 24 h, then incubated with tri-iodothyronine and tetramethylrhodamine-labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin, and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (H) Quantification of bound bacteria corresponding to the images shown in (G) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 6 individual images (×200 magnification) from two coverslips per group]. A representative result of three experiments is shown. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Residue, Expressing, Western Blot, Phospho-proteomics, Membrane, Inhibition, Fluorescence, Incubation, Control, Two Tailed Test, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Bacteria, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Detection of C5a and C5aR1 in human urinary tract. (A–C) C5a and creatinine concentrations in urine from urinary tract infection (UTI) patients and healthy controls. (A) Urinary C5a concentrations. (B) Urinary creatinine concentrations. (C) Ratios of urinary C5a/creatinine concentration. Data were analyzed by Mann–Whitney U two-tailed test. ** P < 0.01, n.s. no significant. (D) C5aR1 expression in human kidney tissue determined by immunochemical staining. Control: negative control, tissue stained with non-specific mouse IgG2a. Scale bar, 50 µm.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Infection, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Expressing, Staining, Control, Negative Control

Journal: PloS one

Article Title: Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin.

doi: 10.1371/journal.pone.0030742

Figure Lengend Snippet: Figure 2. Sphingosine-1-phosphate (S1P) and anaphylatoxin C5a concentrations in plasma and lung tissue. S1P concentrations in plasma (A) or lung tissue lysate (B) or C5a concentrations in plasma (C) or lung tissue lysates (D) from Sphk1+/+ or Sphk12/2 mice were determined before or after i.p. LPS challenge (0.5 mg/kg), using the LC-MS/MS (46) or ELISA techniques. Error bars represent s.d. *p,0.005, **p,0.001, by Student’s t-test. No significant differences in (B, C, D). n = 10 for each genotype and time point. doi:10.1371/journal.pone.0030742.g002

Article Snippet: Cells were stimulated with LPS (500 ng/ml) in the presence or absence of mouse recombinant C5a (R&D Systems) (1 nM).

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: PloS one

Article Title: Sphingosine kinase 1 mediation of expression of the anaphylatoxin receptor C5L2 dampens the inflammatory response to endotoxin.

doi: 10.1371/journal.pone.0030742

Figure Lengend Snippet: Figure 4. Anaphylatoxin C5a-mediated reduction in cytokine and chemokine production depends on Sphk1. BMDMs from Sphk1+/+ or Sphk12/2 mice were stimulated with LPS (500 ng/ml), with LPS concomitant with C5a (1 nM), or without C5a. Tissue culture supernatants were harvested 2 h (TNF-a) or 8 h (IL-6 and KC) after the addition of stimuli. (A) TNF-a; (B); IL-6; (C) KC. Error bars represent s.d. *p,0.05 by Student’s t-test; n = 5 for each genotype; representative for at least three independent experiments. (D) Reduced C5a-induced ERK1/2 and activation. BMDMs from Sphk1+/+ or Sphk12/2 mice were stimulated with C5a (10 nM), LPS (500 ng/ml), LPS and C5a, or saline for 5 min. Cell lysates were processed for immunoblotting with indicated antibodies. Representative of 3 independent experiments showing similar results. UD, undetected. (E) Model: Reduction of inflammatory cytokine production by phagocytes stimulated with C5a requires Sphk1. The model links Sphk1 activity to the cell surface expression of the anaphylatoxin receptor C5L2. LPS, C5a and inflammatory cytokines activate Sphk1 which is required to maintain S1P during inflammation. S1P regulates C5L2 cell surface expression on phagocytes. C5a, via C5L2 expressed on the cell surface, reduces neutrophil inflammation and inflammatory cytokine production by macrophages. doi:10.1371/journal.pone.0030742.g004

Article Snippet: Cells were stimulated with LPS (500 ng/ml) in the presence or absence of mouse recombinant C5a (R&D Systems) (1 nM).

Techniques: Activation Assay, Saline, Western Blot, Activity Assay, Expressing