Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a C4a and C4b restore the astrocytic engulfment of microglial debris in vitro in serum-free culture medium. b Quantifications of the phagocytic influence by complement supplementation and preopsonization in serum-free culture medium. N = 11 independent biological replicates of each group. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). c Scheme of in vivo microglial depletion and time points for analysis. d Reanalysis of RNA-seq data from whole-brain homogenate (GSE108269 ) showing that Gfap and C4b are upregulated and C1qa is downregulated during microglial ablation, whereas C2 , C3 and C4a remain at low levels and are unaffected. N = 5 mice at D0 and N = 4 mice at D2 to D21. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). e qPCR further confirmed the upregulation of C4b in sorted astrocytes upon microglial depletion. N = 5 in each group. Two-tailed independent t test. f Scheme of the in vivo examination of astrocytic engulfment using AAV PHP.eB-based astrocyte labeling and microglial depletion. g Confocal orthogonal colocalization and 3D reconstruction show that C4b −/− impairs the astrocytic engulfment of microglial debris under physiological condition (D21) and upon CSF1R inhibition (D23). h Quantification of microglial debris engulfment by astrocytes. N = 7 (D21) and 8 (D23) WT mice, and N = 3 (D21) and 5 (D23) C4b −/− mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). PLX5622 PLX5622-formulated AIN-76A diet, CD control AIN-76A diet, IV intravenous, MFI mean fluorescence intensity, Ctx cortex, Hipp hippocampus, OB olfactory bulb. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: Recombinant mouse complement component C2 protein CF (C2) was acquired from R&D Systems (Cat#: 6725-SE-010).

Techniques: In Vitro, In Vivo, RNA Sequencing, Two Tailed Test, Labeling, Inhibition, Control, Fluorescence

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: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Extracts from untreated (“-“) or pronase-treated (“+”) 1×10 7 strain B31-e2 spirochetes that ectopically produce the indicated surface lipoproteins were separated by SDS-PAGE and transferred to PVDF membranes. The filters were probed with purified C1 complex (top), C1r enzyme (middle) or C1r proenzyme (bottom), and bound probe revealed by anti-C1r antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. B) Filters prepared identically to panel A were probed with purified C1 complex (top), C1s enzyme (middle) or C1s proenzyme (bottom), and bound probe revealed by anti-C1s antibody, followed by HRP-conjugated anti-mouse antibody. Shown is a representative of 3 experiments. C and D) Biosensors immobilized with GST-ErpB (top) or GST-ErpQ (bottom) were tested by SPR for binding to the indicated concentrations of the enzyme or proenzyme forms of C1r (C) or C1s (D) Injection series were each performed in triplicate. For both panels C) and D), steady-state affinity fits were determined by T200 Biacore Evaluation software and K D values are reported in .

Article Snippet: The next day, membranes were washed in PBS-T and were incubated with α-C1q (Complement Technologies, A200), α-C1r (R&D Systems, MAB1807), or α-C1s (R&D Systems, MAB2060) antibodies, following the manufacturer’s recommended dilutions for western blotting.

Techniques: SDS Page, Purification, Binding Assay, Injection, Software

Journal: bioRxiv

Article Title: Lipoproteome screening of the Lyme disease agent identifies novel inhibitors of antibody-mediated complement killing

doi: 10.1101/2021.09.23.461563

Figure Lengend Snippet: A) Enzymatic cleavage by C1s of the small peptide substrate Z-L-Lys-sBzl was assayed with DTNB (Ellman’s reagent) in the presence of 25 μM BBK32-C (non-inhibitory control) or ErpQ at 25°C for 1hr. Experiments were performed in triplicate. Absorbance was read at 412 nm and signals were normalized to negative control no-substrate wells. B) Top: Proteolytic cleavage of C2 by C1s enzyme produces ∼70kDa C2b and ∼35kDa C2a after 1hr at 37°C. Lanes 1-5: C2b accumulation in the presence (“+”) or absence (“-“) or 25 µM ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 685 nM C2. (Note that the amount of C1s loaded is below the level of detection by SDS-PAGE). Lanes 6-13: C2b accumulation in the presence of 6.25 nM C1s, 685 nM C2 and a two-fold dilution series (from 16 to 0.13 μM) of ErpQ. Bottom: The fraction of C2b relative to total input C2 in the same lane determined by densitometry analysis data are normalized to C2 (lane 5) and C1s digested C2 (lane 6). A representative gel is shown. The experiment was performed three times. C) Top: C4, which consists of 3 polypeptide chains, C4α (97 kDa), C4β (77 kDa), C4γ (33 kDa), is cleaved by C1s enzyme for 1hr at 37 °C to produce C4α’ (88 kDa). Lanes 1-5: SDS-PAGE profile in the presence (“+”) or absence (“-“) or 25 µ M ErpQ, 25 µ M BBK32-C (non-inhibitory control), 6.25 nM C1s, and 616 nM C4. Lanes 6-13: SDS-PAGE profile in the presence of 6.25 nM C1s, 616 nM C4 and a two-fold dilution series (from 25 to 0.20 μM) of ErpQ. Bottom: The fraction of C4α’ relative to input C4β in the same lane and normalized to C1s + C4 positive control (lane 6) and negative control C4 (lane 5) was determined by densitometry analysis.

Article Snippet: The next day, membranes were washed in PBS-T and were incubated with α-C1q (Complement Technologies, A200), α-C1r (R&D Systems, MAB1807), or α-C1s (R&D Systems, MAB2060) antibodies, following the manufacturer’s recommended dilutions for western blotting.

Techniques: Control, Negative Control, SDS Page, 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: 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