human recombinant c3a  (R&D Systems)

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

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: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 1. Anaphylatoxins C3a and C5a differentially regulate endothelial monoculture dnCI in a dose-dependent manner. (A,B) Monitoring of dnCI changes in response to 50 nM, 100 nM and 500 nM of anaphylatoxins C3a and C5a revealed a transient decrease in dnCI by 500 nM C5a after 2 h of treatment in HBMEC. At 24 h after anaphylatoxin stimulation, 500 nM C3a significantly decreased the paracellular resistance in HBMEC compared to the untreated control. (C,D) In HREC, we observed no significant alterations in paracellular resistance after 2 h of C3a or C5a treatment. A dose-dependent increase in dnCI occurred 24 h after treatment initiation in HREC treated with 50 nM C3a, 100 nM C3a, 50 nM C5a and 100 nM C5a compared to the untreated control. (A,C) ↑indicates analyses time points at 2 and 24 h. All data were normalized to the respective cell index (CI) value before the addition of treatment and to the untreated control (dnCI). (A,B) RTCA graph represents data from n = 10 for untreated and 500 nM C3a, n = 6 for 50 nM C3a and 50, 100 and 500 nM C5a and n = 8 for 100 nM C3a treated cells, respectively. (C,D) RTCA graph represents data from n = 9 for untreated, n = 6 for 50 nM C5a and 50, 100 and 500 nM C3a, n = 8 for 100 nM C5a and n = 4 for 500 nM C5a, respectively. (B,D) Two- and twenty-four-hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test was used for datasets including non-parametric data. One-way analysis of variance (ANOVA) with Dunnett’s T3 post hoc test was used for parametric data sets. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 2. C3a and C5a do not change CDH5 gene expression but alter VE-cadherin on protein level in HBMEC and HREC. (A,B) Treatment with 500 nM C3a or 500 nM C5a did not change CDH5 gene expression in HBMEC and HREC compared with the untreated control. (C) Two hours after treatment,

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Gene Expression, Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 3. Anaphylatoxin treatment caused increased C3 expression in HREC and increased C3AR1 expression in HBMEC. (A,B) qRT-PCR analysis revealed no effect of anaphylatoxin treatment on C3 expression in HBMEC but disclosed an increase in C3 expression in 500 nM C5a-treated HREC 2 h post-treatment and a 500 nM C3a and 500 nM C5a mediated increase in C3 gene expression 24 h post-treatment in HREC. (C,D) Two hours’ exposure to 500 nM C5a increased C3AR1 expression in HBMEC, while anaphylatoxin treatment had no significant effect on C3AR1 expression in HREC. (A–D) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four- hour time points were analyzed as separate datasets. Kruskal–Wallis test with Dunn’s multiple comparisons test for datasets including non-parametric data. One-way ANOVA with Dunnett’s T3 post hoc test for parametric datasets. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 4. C3a presence increased in HREC after exposure to C3a and C5a. (A) After 2 h, the C3 protein signal intensity increased in C3a- and C5a-treated HBMEC. The C3a signal in untreated HBMEC exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution. In HREC, C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups after 2 h, but the signal intensity increased in C3a- and C5a-treated HREC. HBMEC and HREC C3a signal intensity remained stable across treatment groups and exhibited a spot-wise pattern across all treatment groups after 2 h. (B) After 24 h, the C3 signal decreased in C3a-treated HBMEC and returned to baseline in C5a-treated cells. In untreated HBMEC, C3a exhibited an uneven distribution pattern, ranging from spot-wise signals to broader distribution 24 h post-treatment initiation. The localization of C3a transitioned from spot-wise distribution to a more diffuse cellular distribution in HBMEC treated with 500 nM C3a and 500 nM C5a 24 h after treatment initiation. HREC C3 protein expression pattern in close proximity to the nucleus remained unchanged across all treatment groups. C3 signal intensity increased in C3a- and C5a-treated HREC after 24 h. The spot-wise C3a distribution pattern seen after 2 h persisted in untreated HREC 24 h after treatment but expanded to a broader, cell-covering signal in cells treated with 500 nM C3a and 500 nMC5a. C3a signal intensity increased in C3a- and C5a-treated HREC 24 h after treatment initiation. (A,B) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. Kruskal-Wallis test for non-parametric data sets, one-way ANOVA with Dunnett’s T3 post hoc test for parametric data sets. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 5. C5 gene and protein expression are elevated in HREC following C5a stimulation, whereas no significant changes are observed in HBMEC. (A,B) qRT-PCR indicated no variations in C5 gene expression among treatment groups or analysis time points at 2 and 24 h in HBMEC. Two hours post-treatment, exposure to 500 nM C3a and 500 nM C5a resulted in a significant upregulation in C5 gene expression in HREC. This effect was reversed 24 h post-treatment. (C,D) Stimulation with 500 nM C3a and 500 nM C5a provoked no change in C5 protein signal strength in HBMEC. Two hours post-treatment, C5 exhibited a spot-wise expression pattern in both untreated and 500 nM C3a-treated HREC, whereas HREC treated with 500 nM C5a showed a broader distribution of immunofluorescent signals. C5a increased C5 signal intensity compared to the untreated control and C3a-treated HREC. After 24 h, C5 detection signal returned to baseline in C3a- and C5a-treated cells. (A,B) Data were collected from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. (C,D) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05, ** p < 0.01.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Control

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 6. C5AR1 transcript expression remains unchanged, but protein detection changes under anaphylatoxic stress. (A,B) qRT-PCR analysis showed no difference in C5AR1 gene expression between untreated and C3a- or C5a-treated HBMEC and HREC. (C) Treatment with 500 nM C3a and 500 nM C5a reduced C5aR1 protein signal in HBMEC after 2 h of treatment. Immunofluorescent staining of C5aR1 in HREC showed a stable and comparable signal after 2 h in all treatment groups. (D) After 24 h, the C5aR1 signal was stable between treatment groups in HBMEC. C5aR1 protein signal intensity increased in 500 nM C5a-treated HREC after 24 h. (A,B) Data were curated from 4 separate cultures as technical replicates. Two- and twenty-four-hour time points were analyzed as separate datasets. One-way ANOVA with Dunnett’s T3 post hoc test. (C,D) Three separate cultures as technical replicates. White boxes visualise magnified areas. Scale bar 50 µm. One-way ANOVA with Dunnett’s T3 post hoc test. * p < 0.05.

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

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

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 7. Adding 50 nM of C5a, but not 100 nM or 500 nM, counteracted the barrier-disruptive effect of 500 nM C3a and improved the HBMEC barrier after 24 h. (A,B) Monitoring of combined anaphylatoxin-treated dnCI changes in HBMEC revealed a C5a dependent increase in dnCI, which counteracted the barrier-disruptive effect of C3a. 24 h post-anaphylatoxin stimulation, HBMEC treated with 500 nM C3a + 50 nM C5a reached a significantly higher dnCI compared to 500 nM C3a- treated cells. (C,D) RTCA measurement revealed an increase in dnCI in HREC treated with 500 nM C3a + 100 nM C5a 2 h after treatment addition compared to 500 nM C3a-treated cells. This regulation was maintained for 24 h after exposure to treatment. (E) Immunocytochemistry revealed a homoge- neous endothelial monolayer in all treatment groups in HBMEC, 2 h post-treatment. (F) Twenty-four hours post-treatment, 500 nM C3a decreased the VE-cadherin signal intensity, whereas treatment with 500 nM C3a + 50 nM C5a obtained a VE-cadherin signal strength similar to that of the untreated

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Immunocytochemistry

Journal: International journal of molecular sciences

Article Title: C3a Mediates Endothelial Barrier Disruption in Brain-Derived, but Not Retinal, Human Endothelial Cells.

doi: 10.3390/ijms252011240

Figure Lengend Snippet: Figure 8. 500 nM C3a and C5a increase the transcellular permeability for IgG in HBMEC but not in HREC, reflecting reduced cell–cell contact integrity at 500 nM C3a but not enhanced paracellular

Article Snippet: Following a 17 h growth phase, HBMEC and HREC were subjected to recombinant C3a (R&D Systems, Minneapolis, MN, USA, #3677-C3-025) or recombinant C5a (R&D Systems, Minneapolis, MN, USA, #2037-C5-025/CF) treatment for 2 and 24 h. Both anaphylatoxins were diluted in the respective culture medium.

Techniques: Permeability

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 2: Te concentrations of complement pyrolysis products were found higher in pleural efusion than in plasma from TPE patients. (a) Complement pyrolysis products, including C3a, C3b, C3d, C5a, and opsonin receptors (CR1 and CR3) were detected in human tuberculosis pleural biopsy samples by immunohistochemistry. (Original magnifcation, ×200) (n 4). (b) Higher levels of complement pyrolysis products were found in pleural efusion than in plasma in TPE patients (n 20). Te concentrations of complement pyrolysis products in pleural fuid and plasma from TPE patients were measured by ELISA (n 20).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Clinical Proteomics, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 3: Monocyte migration was inhibited by antibodies that blocked CXCL12. (a) CXCL12 and CXCR4 staining by immunohisto- chemistry in human pleural biopsy (original magnifcation, ×200) (n 4). (b) Te concentration of CXCL12 in pleural fuid and plasma from TPE patients was measured by ELISA (n 20). (c) CXCL12 produced by PMCs was measured by PCR and ELISA after Mpt64 and anaphylatoxin activation. PMCs were incubated for 24 hours in control media or in media with Mpt64 (20 μg/ml) and with or without human C3a (100 nM) or C3aRA (100 nM) (n 4). (d) Coexpression of CXCL12-CXCR4 in PMCs and monocytes from TPE was detected by immunofuorescence (original magnifcation, ×400) (n 4). (e) Monocytes were seeded into the top chamber of a transwell system, and the supernatant from PMCs cultured with anti-CXCL12 antibody or PBS were placed in the bottom chamber. Te migratory index was calculated by dividing the number of monocytes that migrated in response to the supernatants from cultured PMCs by the number of monocytes that migrated in response to the control. ∗vs the MO-PBS group, ∗∗P < 0.01. #vs the MO-PMC group, ##P < 0.01 (n 4).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Migration, Staining, Immunohistochemistry, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Produced, Activation Assay, Incubation, Control, Cell Culture

Journal: JCI insight

Article Title: C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy.

doi: 10.1172/jci.insight.172976

Figure Lengend Snippet: Figure 2. C3aR/C3aR signaling plays a key role in PLA2R+ MN-induced injury. (A) Representative picture of immunofluorescence staining confirming expres- sion of C3aR (red, top panel) in podocytes in vitro. DAPI nuclear staining: blue. Magnification, ×20. (B) Box plot graph of fluorescein absorbance (485 nm/535 nm, 0.1 second) in filtrate collected from channel F 60 minutes after addition of albumin. Exposure of GOAC to 50 nM C3a for 24 hours increased albumin leak- age compared with the control group. Exposure to the chip to 50 nM C3aR antagonist was able to prevent the deleterious C3a effect, confirming the role of C3a in driving injury in the GOAC. *P < 0.05, n ≥ 4/group. (C) Box plot graph of fluorescein absorbance (485 nm/535 nm, 0.1 second) in filtrate collected from channel F 60 minutes after addition of albumin. Addition of 50 nM C3aR antagonist to GOAC was able to prevent leakage caused by MN serum, further confirming the involvement of C3a in podocyte damage. ****P < 0.0001, n ≥ 4/group. (D) Measurement of C3ar RNA expression by qPCR in control hAKPC-derived podocytes and hAKPC-derived podocytes in which C3ar was knocked down, confirming an 80% decrease in its expression. n = 3, Ctrl hAKPC; n = 3, KD hAKPC. (E) Western blotting analysis of C3aR in hAKPC-derived podocytes and hAKPC-derived podocytes in which C3ar was knocked down confirmed an efficient decrease in protein expression. Measured density for C3aR bands (54 KDa) was normalized against β-actin, showing a significantly decreased protein level. ***P < 0.001. β-Actin: 42 KDa; n = 3/group. (F) Box plot graph of fluorescein absorbance (485 nm/535 nm, 0.1 second) in filtrate collected from channel F 60 minutes after addition of albumin confirming a statistically significant decrease in MN sera–induced albumin leakage in GOAC generated with hAKPCC3aR KD podocytes compared with control. ****P < 0.0001; n ≥ 4/group. All statistical values determined by 1-way ANOVA, with the exclusion of E , which used 2-tailed Student’s t test.

Article Snippet: Exposure of GOAC to recombinant C3a, C5a, C3aR, and C5aR antagonist We exposed GOAC to 50 nM C3a (3677-C3-025, R&D) or C3a + C3aR antagonist (C3aRA, SB290157, Cayman Chemical) in GOAC culture media for 24 hours as previously described (13, 15, 21).

Techniques: Immunofluorescence, Staining, In Vitro, Control, Albumin Addition, RNA Expression, Derivative Assay, Expressing, Western Blot, Generated

Journal: JCI insight

Article Title: C3aR-initiated signaling is a critical mechanism of podocyte injury in membranous nephropathy.

doi: 10.1172/jci.insight.172976

Figure Lengend Snippet: Figure 5. DAF expression and C3a/C3aR signaling affect disease severity in murine anti-THSD7a antibody–induced MN. (A) Box plot graph of fluorescein absorbance (485 nm/535 nm, 0.1 second) in filtrate collected from channel F 60 minutes after addition of albumin, confirming a statistically significant increase in anti-THSD7A+ sera–induced albumin leakage in GOAC compared with control. Statistical values determined by 1-way ANOVA. *P < 0.05; **P < 0.01; n ≥ 4/ group. (B) C3b and synaptopodin staining in representative glomeruli from WT and DAF–/– mice at 8 weeks after injection of serum from patients with MN with anti-THSD7a antibodies. Scale bar: 50 μm. (C) Quantification of C3b deposition (BALB/c WT, n = 3; DAF–/–, n = 3). Glomerular fluorescence intensity was quanti- fied measuring MFI in all glomeruli of a kidney section. DAF and C3b expression was then divided by the average MFI of glomeruli stained with isotype (normal- ized data) using ImageJ software. Statistical values determined by Mann-Whitney U test. ****P < 0.0001. (D) DAF and synaptopodin staining in representative glomeruli from WT mice at 0, 2, and 8 weeks after injection of serum from patients with MN with anti-THSD7a antibodies. Scale bar: 50 μm. (E) Quantification of DAF immunofluorescence (0 weeks, n = 3; 2 weeks, n = 2; 8 weeks, n = 2). Glomerular fluorescence intensity was quantified relatively to isotype using ImageJ software. Statistical values determined by Kruskal-Wallis test. ****P < 0.0001. (F) Urinary albumin/creatinine (ACR) levels in DAF–/– BALB/c male mice injected with serum from patients with MN with anti-THSD7a antibodies. One group of mice was treated with C3aR antagonist starting from the day of serum injection (THSD7a sera, n = 3; THSD7a sera + C3aRA, n = 3). Statistical values determined by 2-way ANOVA. *P < 0.05, **P < 0.001.

Article Snippet: Exposure of GOAC to recombinant C3a, C5a, C3aR, and C5aR antagonist We exposed GOAC to 50 nM C3a (3677-C3-025, R&D) or C3a + C3aR antagonist (C3aRA, SB290157, Cayman Chemical) in GOAC culture media for 24 hours as previously described (13, 15, 21).

Techniques: Expressing, Control, Staining, Injection, Fluorescence, Software, MANN-WHITNEY, Immunofluorescence