human recombinant c3a  (R&D Systems)

Journal: Cells

Article Title: House Dust Mite Nebulization Drives Alarmin and Complement Activation in a Murine Tracheal Air–Liquid Interface Culture System

doi: 10.3390/cells14201598

Figure Lengend Snippet: Time-dependent complement activation and anaphylatoxin receptor expression in ALI cultures exposed to HDM. ( a ) C3 and C3a concentrations in cell lysates and the SN of unstimulated (steady-state) ALI cultures and 24, 48 and 72 h after HDM exposure (steady-state, 24, 48 and 72 h: n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001. ( b ) HDM and HI-HDM mediated cleavage of hC3 into hC3a ( n = 3 independent experiments). Data are shown as mean ± SEM. Data were analyzed using an unpaired T -test. *** p < 0.001. ( c ) C5 and C5a concentrations in cell lysates and the SN of steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). ( d ) Comparison of C3 and C5 ( left panel ), C3a and C5a ( right panel ) concentrations in cell lysates from steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. **** p < 0.0001. ( e ) Immunofluorescence analysis of C3aR and C5aR1 expression in unstimulated ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM ( n = 3). Data were analyzed using one-way ANOVA with Holm–Šidák’s multiple comparisons test. * p < 0.05; ** p < 0.01. The number of experiments shown in ( a , c – e ) refers to biological replicates derived from 6 independent preparations of trachea, each representing a separate cell isolation and culture.

Article Snippet: Plates were washed four times with 0.05% Tween 20 in PBS; serial dilutions of recombinant mouse C3a (R&D Systems #8085-C3-025—Minneapolis, MN, USA) were performed; and samples were added (25 μL/well) and incubated at RT for 90 min. After washing four times with 0.05% Tween 20 in PBS, 25 μL/well of 1 μg/mL in 1% BSA/PBS biotinylated anti-mouse C3a detection antibody (clone I87-419, BD Biosciences #558251—San Jose, CA, USA) was added and incubated for 1 h at RT.

Techniques: Activation Assay, Expressing, Comparison, Immunofluorescence, Derivative Assay, Cell Isolation

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) The blood coagulation cascade, highlighting molecules whose levels in urine are significantly correlated with renal pathology AI (blue font), CI (CI) (red font), or both (purple font) in LN. Other proteins are listed in black font (interrogated but not significantly changed) or grey font (not interrogated by the proteomic screen). Uninterrupted arrows indicate activation, and interrupted arrows signify inhibition or cleavage of downstream protein or substrate. The yellow bubbles highlight only proteins significantly elevated in LN with high-CI versus low-CI (FC ≥ 2; P < 0.05) or higher in CI than in AI by at least 10%. The pink bubbles highlight proteins whose levels were significantly elevated only in patients with LN with high-AI versus low-AI (FC ≥ 2; P < 0.05) or higher in AI than in CI by at least 10% (in terms of correlation coefficient or FC). Also shown is a Spearman correlation heatmap displaying correlations among the 27 coagulation-related proteins and renal pathology metrics. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. ( B ) The complement activation pathway highlighting molecules whose levels in urine are significantly elevated with AI, CI, or both. See A for other annotation details. Also shown is a Spearman correlation heatmap displaying correlations among the 32 complement related proteins and their paired renal pathology metrics, as detailed in A . α2-AP, MG, α2-antiplasmin, α2-macroglobulin; APC, activated protein C; AT, antithrombin; B, factor B; BK, bradykinin; C1 INH, C1 esterase inhibitor; C4BP, C4 binding protein; CL-K1, collectin kidney 1; D, factor D; DAF, decay-accelerating factor; FDP, fibrin degradation products; H, factor H; I, factor I; MAP-1, MBL/ficolin-associated protein 1; MASP, mannan-binding lectin-associated serine protease; MBL, mannose-binding lectin; MCP, membrane cofactor protein; P, properdin; PK, prekallikrein; sMAP, small MBL-associated protein; TM, thrombomodulin; tPA, tissue plasminogen activator; uPA, urokinase.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Coagulation, Activation Assay, Inhibition, Binding Assay, Membrane

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: ( A ) Shown are representative fields from 3 independent experiments. Complement proteins C3a and C5a increased the expression of ECM proteins in THP1 macrophages, BMDMs, and HK2 proximal tubule cells after 72 hours of treatment in serum-free medium. Scale bars: 50 μm. ( B – D ) The scatter plots show the mean staining intensity per THP-1 macrophage ( B ), BMDM ( C ), and HK2 proximal tubule cell ( D ), normalized to expression levels in their respective vehicle-treated groups. Each data point corresponds to quantified fluorescence intensity in a single field of view (FOV) from the microscope, and the larger dots represent the average of FOVs in biological replicates, each of which is color coded. RT-qPCR analysis of ECM protein coding genes were measured in BMDMs ( E ) and in HK2 proximal tubule cells ( F ). Gene expression was normalized to the expression of 18S ribosomal RNA in the same sample and then normalized to the expression level of vehicle-treated group ( n = 4). * P < 0.05, ** P < 0.01, and *** P < 0.001 by 2-tailed Student’s t test.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Expressing, Staining, Fluorescence, Microscopy, Quantitative RT-PCR, Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: Urine proteins reveal distinct coagulation and complement cascades underlying acute versus chronic lupus nephritis

doi: 10.1172/JCI186143

Figure Lengend Snippet: Circulating immune complexes and Abs planted directly within glomerular and tubulo-interstitial regions of the kidneys may fix complement, resulting in complement activation. The alternative pathway may further amplify complement activation within the kidneys. The products of C3 and C5 convertases, including the anaphylatoxins C3a and C5a, engage cognate receptors on a wide spectrum of immune cells, leading to immune cell activation, release of cytokines and chemokines, and acute inflammation, leading to high AI, as depicted on the left. Long-standing, unresolved complement activation and eventual formation of MAC may additionally engage and activate more immune and renal-resident cells, leading to tissue damage and repair, ECM deposition, and renal fibrosis, leading to high CI, as depicted on the right.

Article Snippet: After 3 days of differentiation, the medium was replaced with serum-free medium for 24 hours, after which the cells were treated for 72 hours with either vehicle or 10 ng/mL C3a (R&D Systems 3677-C3-025) or 10 ng/mL of C5a (R&D Systems 2037-C5-025/CF).

Techniques: Activation Assay

Journal: Computational and structural biotechnology journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer.

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Fig. 8. C3aR antagonist (SB290157) attenuated the effects of C3a activation in pancreatic cancer. (A) Representative images of EdU assays showed that the treatment of SB290157 attenuated C3a-induced proliferation in Panc-1 cells. (B) The bar plot showed the percentage of EdU+ cells per field. (C) Representative images of Transwell assays showed that the treatment of SB290157 attenuated C3a-induced migration in Panc-1 cells. (D) The bar plot showed the percentage of migrated cells per field. (E) CCK-8 assay showed that the treatment of SB290157 attenuated C3a-induced gemcitabine resistance in Panc-1 cells. F-K A mouse subcutaneous tumor model of Panc-1 cells was constructed. The mice were randomly grouped and treated with vehicle, 20 mg/kg gemcitabine, 20 mg/kg SB290157, 20 mg/kg gem- citabine combined with 20 mg/kg SB29015 by intraperitoneal injection once a day. (F) Line charts of volume changes of subcutaneous tumor. (G) On the 14th day of treatment, subcutaneous tumors were separated to show tumor size. Frozen sections were prepared from tumor tissues. IF staining with antibodies to Ki-67 (in red color, (H)) and CC3 (in red color, (J)) was performed to detect proliferation and apoptosis of Panc-1 cells. And the percentage of Ki-67+ cells (I) or CC3+ cells (K) per field was shown by bar plots. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Migration, CCK-8 Assay, Construct, Injection, Staining