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: medRxiv

Article Title: Differential virulence potential of different clades of multidrug-resistant Klebsiella pneumoniae ST258

doi: 10.64898/2026.03.28.26349612

Figure Lengend Snippet: (A and B) Bacteria recovered after 30-minute exposure of 10 8 CFU/mL to 85% human serum. Limit of detection (LOD) is indicated with a dashed line. (A) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (B) 6 biological replicates per strain were tested. (C and D) C3 binding to bacteria was measured using an indirect ELISA. (C) 13 Clade 1 isolates and 36 Clade 2 isolates were tested, each with at least two biological replicates. (D) 6 biological replicates were tested per strain, each with at least 5 technical replicates. (E) Correlation between C3 binding and serum survival of Clade 2 isolates was assessed using Spearman correlation. (F) Uronic acid was quantified from a selection of Clade 1 (n=6) and Clade 2 (n=10) isolates with 3 biological replicates per isolate. Significance was assessed with Welch’s t-test. (G) Correlation between uronic acid quantity and serum survival of 10 Clade 2 isolates was assessed using Spearman correlation. (H) OD-normalized bacteria were reacted with 5% human serum in PBS for 30 minutes. C5a from the supernatant of the reaction was quantified using a sandwich ELISA. At least two biological replicates per isolate were performed. For panels A-D and H, P ≤ 0.05: *, P ≤ 0.01: **, P ≤ 0.001: *** by Mann-Whitney test.

Article Snippet: C5a quantification was performed using the Human Complement Component C5a DuoSet ELISA from R&D systems.

Techniques: Bacteria, Binding Assay, Indirect ELISA, Selection, Sandwich ELISA, MANN-WHITNEY

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Library size-normalized counts detected by RNA sequencing in mouse RPM and in M0-differentiated BMDM (3 mice each; only GPCRs with average count >15 displayed). B Gprc5b expression in RPM and lymph node-derived lymphocytes was determined by NanoString RNA analysis (cells pooled from two mice per data point). C Knockout efficiency in RPM from control mice (white) and M-G5b-KOs (gray) was analyzed by qRT-PCR (C, n = 4, data normalized to Gapdh and controls set to 1). D Knockout efficiency in RPM was analyzed by immunoblotting (unspecific band around 38 kDa; the higher of the two specific bands probably represents glycosylated GPRC5B ; GAPDH as loading control). E, F Expression of Nos2 (E) and Tnf (F) was determined in RPM by qRT-PCR under basal conditions and after 6 h of stimulation with 1 µg/ml LPS ( n = 11/12/12/12 in E, 12/10/11/12 in F), data normalized to Gapdh and control set to 1). G Basal and C5a (20 ng/ml)-induced transwell migration in RPM (all cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration) ( n = 3). H The distance travelled by individual RPM in response to different chemotactic factors (C5a, 20 nM; CCL5, 10 ng/ml; fMLP, 10 nM) was determined by live cell imaging ( n = 512 cells from 2 mice per group; cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration, arb. units: arbitrary units). Phagocytic activity of LPS (1 µg/ml, 6 h)-stimulated RPM was determined by uptake of pHrodo E. coli bioparticles ( I , J , n = 5) or pHrodo-labeled apoptotic thymocytes ( K , L n = 10); I + K show original traces, J + L statistical evaluation of areas under the curve (AUC). Body weight change ( M ) and bacterial colony-forming units in peritoneal lavage fluid harvested 24 h after injection of fecal bacteria ( N ) ( n = 10). O, P Numbers of CD11b + , F4/80 + , MHCII - , Tim4 + RPM and CD11b + , F4/80 lo , MHCII + , CCR2 + BMDM before and 3, 24, and 54 h after i.p. injection of fecal bacteria ( n = 3/3/3/3/11/12/5/5). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t -test (C, J, L, N), two-way ANOVA (E-H) or two-way RM-ANOVA (M) with Sidak’s multiple comparison test, unpaired two-sided t-test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (O, P). * P < 0.05; *** P < 0.001; **** P < 0.0001; n , number of individual mice. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Knock-Out, Control, Quantitative RT-PCR, Western Blot, Migration, Live Cell Imaging, Activity Assay, Labeling, Injection, Bacteria, Comparison

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Knockout efficiency was determined by qRT-PCR in RPM and M0 BMDM (data normalized to Gapdh and RPM controls set to 1) ( n = 12). Analyses in resting and LPS (1 μg/ml, 6 h)-stimulated M0 BMDM: Expression of inflammatory genes ( B , C ; n = 15/14/15/15 in B, 15/14/15/15 in C), production of NOx ( D ; n = 3) or release of cytokines ( E , n = 3). F Transwell migration of M1 BMDM in response to different chemotactic factors ( n = 6) (CCL5: 75 ng/ml, CCL2: 10 ng/ml, SDF-1β: 100 ng/ml, C5a: 20 ng/ml, fMLP: 10 nM). Uptake of pHrodo E.coli fragments by M0 BMDM: G , exemplary curves; H , statistical analysis of AUC ( n = 6). I Flow cytometric analysis of CD11b-positive cells in the combined infarct and border zones of hearts harvested 4 days after infarction ( n = 5). J Echocardiographic analysis of ejection fraction (EF%) before and after infarction (8 controls, 4 KOs). K Histological analysis of scar size in hearts harvested 21 days after infarction ( n = 7 controls, 4 KOs), left ventricle (LV). L, M DSS colitis: Disease activity index integrating body weight change, stool consistency, intestinal bleeding (L) and colon length on day 6 (M) ( n = 7(L), 7/7/10/11 in M). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t- test (A, H, K), two-way ANOVA with Sidak’s multiple comparisons test (B-F, J, M), unpaired two-sided t -test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (I), two-way repeated measures ANOVA with Sidak’s multiple comparisons test (L). n, number of mice per group; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Migration, Activity Assay

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

Article Title: Complement as Prognostic Biomarker and Potential Therapeutic Target in Renal Cell Carcinoma.

doi: 10.4049/jimmunol.2000511

Figure Lengend Snippet: FIGURE 5. Complement in mouse Renca model of RCC. (A) C3 deposition along CD31 Ab-stained vasculature, (B) C1q and C3, (C) C5a concentration in plasma from tumor-free (TF) and tumor-bearing (TB) mice, *p , 0.0001 by t test. (D) C1q and IgM. (E) Annexin Vand IgM. (F) C5aR1 and CD11b. (G) C5aR1 and CD8+. Arrows denote areas of colocalization. Scale bar, 50 mm. (A, B, and D–G) immunofluorescence and (C) ELISA.

Article Snippet: ELISA of human and mouse plasma Mouse C5a ELISA was performed according to the manufacturer’s instructions (DY2150; R&D Systems).

Techniques: Staining, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cellular and molecular life sciences : CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Fig. 2 C5a elicited nephrotic syndrome like manifestation in mice. ICR mice were injected with 200 ng recombinant C5a (10 lg/kg) every 3 days through tail vein. Urinary albumin/ creatinine (A/C) ratio (a), serum albumin (b), triglyceride (c), and total cholesterol (d) levels were monitored every other day in the experimental group (filled square) and control mice which were injected with normal saline (open square). N = 5 for each group. The data shown represent mean ± SD. *P \ 0.05; **P \ 0.001

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 lg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Injection, Recombinant, Control, Saline

Journal: Cellular and molecular life sciences : CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Fig. 5 C5a-induced disassembly and redistribution of adherens junctional proteins. Mouse KECs were grown to confluency. The culture medium was replaced with M199 medium for serum fasting overnight before the indicated concentration of C5a (ng/ml) was applied to the cells for 60 min. After phase contrast images of KECs were acquired with CCD camera (a), the KECs were processed for immunofluorescence using mouse anti-VE cadherin (b) and rabbit anti-b-catenin (c) antibodies. The actin cytoskeleton organization was revealed by TRITC-rhodamine labeled phalloidin to stain for polymerized actin (d). Scale bars 10 lm

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 lg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Concentration Assay, Labeling, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Fig. 6 C5a-induced cellular contraction, myosin contraction and actin stress fiber formation of KECs in a ROCK dependent way. a KECs were serum fasted overnight in M199 medium before C5a (50 ng/ml) was applied to the cells for 60 min with (right) or without (left) 10 lM Y27632 pretreatment for 30 min before phase contrast images were taken. b The culture condition was the same as in (a) except the cells were maintained at a confluent density before staining with non-muscle myosin II heavy chain B antibody (MYHb, red) and FITC-labeled phalloidin (green). c C5a effect on the actin cytoskeleton in vivo was shown. The intensive phalloidin staining in microvilli of the surrounding renal tubules (arrowheads) serves as a reference point to compare the intensities of phalloidin staining in the glomeruli (arrows) on fresh frozen renal sections from control or C5a (50 ng/ml) treated mice. d Confocal sectioning result following immunofluorescence study using VE-cadherin labeling on fresh frozen renal sections from control or C5a (50 ng/ml) treated mice was shown. Scale bars 10 lm

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 lg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Staining, Labeling, In Vivo, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Fig. 7 C5a-induced RhoA/ ROCK1-dependent myosin light chain activation which led to an increase in the permeability of KECs. KECs were serum fasted in M199 medium for 3 h. Cells were further incubated for 60 min in the presence of the indicated concentration of recombinant C5a before cell lysates were harvested for Rho activation assay (a), Western blotting analysis using the antibodies recognizing total and phospho-specific ROCK1 (b), and total myosin light chain (MLC) and phosphoserine- specific MLC at the amino acid position 19 (c). Y, 10 lM Y27632; PTX, 100 ng/ml pertussis toxin pre-treated for 30 min. (d) Renal glomeruli were isolated from mice treated with 50 ng/ml recombinant C5a as the procedure described in the ‘‘Materials and methods’’ when the proteinuria was first evident. Cell lysates were prepared in NP-40 buffer for RhoA activation assay. e, f KECs were cultured on Transwell plates and pretreated with the indicated inhibitors for 30 min. Then, 50 ng/ml recombinant C5a was applied into the transwell plates for an additional 60 min before permeability assays were performed. The data shown represent mean ± SD (N = 4). *P \ 0.05; **P \ 0.001

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 lg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Activation Assay, Permeability, Incubation, Concentration Assay, Recombinant, Western Blot, Isolation, Cell Culture

Journal: Cellular and molecular life sciences : CMLS

Article Title: Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

doi: 10.1007/s00018-015-1888-0

Figure Lengend Snippet: Fig. 8 Y27632 inhibited the C5a-elicited proteinuria in a murine model. ICR mice were injected with 200 ng recombinant C5a (10 lg/kg) every 3 days through tail vein for 21 days, and Y27632 were administrated 0.6 mg (30 mg/ kg) orally once daily since day 6. Urinary albumin/creatinine ratio (a), serum albumin (b), triglyceride (c), and total cholesterol (d) levels were monitored every 3 days. The data shown represent mean ± SD for each treatment group (N = 5). *P \ 0.05

Article Snippet: The ICR mice were treated with normal saline as the control or recombinant mouse C5a (R&D Systems), respectively, with the dose of 10 lg/kg (200 ng per mouse) every 3 days through tail vein injection.

Techniques: Injection, Recombinant