Journal: Journal of Clinical Investigation

Article Title: Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

doi: 10.1172/jci140966

Figure Lengend Snippet: Figure 1. C3a/C3aR signaling regulates age-associated endothelial VCAM1 expression. (A) ELISA measurement of C3 levels in WT mouse brain lysates at 2, 12, and 20 months (n = 8/group). (B) Immunofluorescence staining using anti-GFAP and anti-C3 antibodies demonstrated localization of C3 to astrocytes. (C) Quantification confirming increased C3 staining within GFAP + astrocytes in the hippocampus with age (n = 5/age). (D) Triple immunostaining of isolated vessels from WT and C3ar1 –/– brains using anti-GFAP, anti–VE-cadherin, and anti-C3aR antibodies showing positive C3aR staining along endothelial cell surface that was not present in C3ar1–/– vessels. (E) Triple immunostaining of brain tissue with anti-Glut1, anti-C3aR, and anti-CD31 or anti–PDGFR-β, anti-C3aR, and anti–Col IV, demonstrating expression of C3aR on brain endothelial cells but not pericytes. (F and G) Immunofluorescence staining and quantification using anti-CD31 and anti-VCAM1 antibodies of WT or C3ar1–/– mouse cortices at 2, 12, and 20 months demonstrated an increase in VCAM1 with age in WT mice, but VCAM1 was rescued in the absence of C3aR. All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 20 μm (B), 10 μm (D), 15 μm (E), and 50 μm (F).

Article Snippet: Confluent cells were treated with IL-1β (10 ng/mL, R&D Systems, 201-LB-005), C3a (500 nM, R&D Systems, 3677-C3-025), C5a (250 nM, R&D Systems, 2037-C5-025), ionomycin (10 μM, Cayman Chemical, 10004974), or in combination with one of the inhibitors SB290157 (5 μM, Calbiochem, 559410), W7 (50 μM, Tocris, 0369) or BAPTA-AM (1 μM, Tocris, 2787).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Triple Immunostaining, Isolation

Journal: Cellular & molecular medicine: open access

Article Title: Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

doi:

Figure Lengend Snippet: RNA interference suppresses local complement activation. BALF collected from Figure 1 were analyzed for C3a (A) and C5a (B) levels in the lung by ELISA. Values: Means ± SEM. (n=5-7 per group). One-way ANOVA, Newman-Keuls (A, B). Compared to bleomycin: ***: p<0.001; **: p<0.01; *: p<0.05. Results are representative of three independent experiments.

Article Snippet: These studies used recombinant human C3a and C5a (100 nM; Complement Technology, Inc., Tyler, TX), platelet-derived TGF-β1 (2 ng/ml; Roche Diagnostics, Germany).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular & molecular medicine: open access

Article Title: Potential Mechanisms Underlying TGF-β-mediated Complement Activation in Lung Fibrosis

doi:

Figure Lengend Snippet: miRNA regulation in response to TGF-β, C3a and C5a for 24 h in normal primary human SAECs.

Article Snippet: These studies used recombinant human C3a and C5a (100 nM; Complement Technology, Inc., Tyler, TX), platelet-derived TGF-β1 (2 ng/ml; Roche Diagnostics, Germany).

Techniques:

Journal: Oncoimmunology

Article Title: Blocking C5aR signaling promotes the anti-tumor efficacy of PD-1/PD-L1 blockade

doi: 10.1080/2162402X.2017.1349587

Figure Lengend Snippet: Anti-PD-L1 treatment leads to activation of the complement system. MC38 tumor-bearing mice (∼100 mm3) were intraperitoneally injected with anti-PD-L1 antibodies. The tumors were removed at the indicated time points (n = 5–6 mice/group). (A) C3a and C5a levels in homogenates from tumors and spleens were quantified by ELISA. (B) C1q and C3b/iC3b/C3c staining of tumors and spleens were quantified by IHC. (C) C3b/iC3b/C3c levels of homogenates from tumors and spleens were quantified by ELISA. One-way ANOVA (A) and Multiple t-test (C) were used to evaluate statistical significance (*p < 0.05, **p < 0.01).

Article Snippet: Biotinylated Rat Anti-Mouse C3a (clone I87–419, BD, 2 μg/ml) was used for detection, and purified recombinant mouse C3a (BD) was used as a standard.

Techniques: Activation Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining