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: 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 role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: scRNA-seq Analysis of Complement C5 Components and Intercellular Communication in Retinal Detachment. (A) UMAP feature plots (left) show C5, C5AR1, and C5AR2 expression across retinal cell clusters in RRD and RRDCD, with color intensity indicating expression levels. Violin plots (right) compare cell type-specific expression distributions between conditions. (B) CellPhoneDB-derived dot plot displays ligand-receptor interactions, with bubble size reflecting interaction significance (-log₁₀ p-value) and color denoting mean expression. Comparisons include C5-C5AR1, C3-C5AR2, TNF-TNFRSF1A/B, and VEGF (VEGFA-FLT1, VEGFB-NRP1) pathways between RRD and RRDCD.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: Expressing, Derivative Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: The role of microglia and complement C5/C5a in the pathogenesis of rhegmatogenous retinal detachment with choroidal detachment

doi: 10.1016/j.csbj.2025.08.019

Figure Lengend Snippet: Characterization of complement C5 effects on cellular responses and molecular markers. (A) MTT assay assessing the viability/metabolic activity of RF/6 A choroidal vascular endothelial cells after 24-hour co-culture with primary mouse retinal microglia that were pre-treated with varying concentrations of complement C5 (0, 0.5, 1.0, 2.5 µg/mL)， with results expressed as ODsample/ODblank ratios. (B) Apoptotic effects of C5-treated (1 μg/mL, 24 h) microglia on RF/6 A choroidal vascular endothelial cells, as evidenced by TUNEL staining (red) and quantification of TUNEL-positive area. (C) C5 exposure (1 μg/mL, 24 h) altered microglial cell viability (MTT assay, Student's t -test) and morphology (brightfield images). (D) ZO-1 immunofluorescence (red) in ARPE-19 cells co-cultured with C5-treated retinal microglia (1 μg/mL, 24 h) showed tight junction integrity (nuclei: DAPI, blue). (E) CD86 expression (red) increased in retinal primary mouse microglia following C5 treatment (1 μg/mL, 24 h). (F) Western blot confirmed elevated CD86 protein levels in C5-treated microglia (1 μg/mL, 24 h). (G) ELISA detected significantly higher TNF-α and IL-1β secretion in C5-treated microglia (1 μg/mL, 24 h). Data represent mean ± SD of ≥ 3 independent experiments.

Article Snippet: Experimental groups were treated with complement C5 (HY-P7695; MedChemExpress) at concentrations of 0.5 μg/mL, 1.0 μg/mL, and 2.5 μg/mL, while the control group received no complement C5.

Techniques: MTT Assay, Activity Assay, Co-Culture Assay, TUNEL Assay, Staining, Immunofluorescence, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 1. Ca2+ influx after stimulation of PMN cells with C5a is required for efficient chemo- taxis. (A, B) PMN cells from C57Bl/6 mice were loaded with Fura2/AM and stimulated or not (w/o C5a) with either C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer (C5a) or in buffer sup- plemented with EGTA (0.5 mM) (EGTA + C5a). (A) One representative measurement of n = 4 spectrofluorometric measurements are pre- sented. (B) In the same setting, maximal ([Ca2+]i ± SEM) values were calculated of n = 4 spectrofluorometric measurements per group; *P < 0.05, determined by 2-tailed Stu- dent´s t test. (C) PMN cells from C57Bl/6 mice were incubated with a Ca2+-specific chelator, EGTA and assayed for efficient C5a (50 ng/ml)- elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each per- formed in duplicate; **P < 0.01, determined by 2-tailed Student´s t test.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Incubation, Chemotaxis Assay, Migration

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 2. Differential involvement of Orai1 and STIM1 in C5a-induced Ca2+ influx and SOCE in PMN. (A, C) Orai1−/−or Stim1−/−PMN cells loaded with Fura2/AM and stimulated with C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer. One representative spec- trofluorometric measurement of n = 4 measure- ments and maximal ([Ca2+]i ± SEM) values are presented (*P < 0.05, determined by 2-tailed Stu- dent´s t test). (B, D) Orai1−/−or Stim1−/−PMN cells loaded with Fura2/AM and treated with Tg (2 μM) in EGTA-containing buffer (w/o [Ca2+]e) followed by the addition of CaCl2 (1 mM [Ca2+]e) and monitoring of [Ca2+]i. Representative spectrofluorometric measure- ments and maximal ([Ca2+]i ± SEM) values (n = 4 per group, in the presence of 1 mM CaCl2 in measuring buffer are shown) (**P < 0.01, determined by 2-tailed Student´s t test).

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques:

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 3. Orai1 is required for efficient C5a- elicited chemotaxis of PMN in vitro and peri- toneal recruitment in vivo. (A) PMN cells from Stim1−/−or Orai1−/−and matched wild- type (Stim1+/+ and Orai1+/+) BM chimeras were assayed for efficient C5a (50 ng/ml)-elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (**P < 0.01, determined by 2-tailed Student´s t test). (B) Orai1−/−or Orai1+/+ BM chimeras were injected with 200μl of C5a (1 μg/ml) or 200 μl of PBS. Two hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4-5 mice per group (*P < 0.05, deter- mined by 2-tailed Student´s t test). Groups of 2– 3 Orai1−/−BM chimeras and two matched wild- type mice were assayed together. (C) Orai1−/−or Orai1+/+ Gr1-positive PMN were characterized for C5aR expression by flow cytometry. Rep- resentative histogram (left) and mean fluores- cence intensity (MFI) ± SEM of n = 3 measure- ments (right) are depicted. P: ns = non signifi- cant, determined by 2-tailed Student´s t test.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Chemotaxis Assay, In Vitro, In Vivo, Migration, Injection, Expressing, Cytometry

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 4. The requirement of extracellular Ca2+ and Orai1 for efficient PMN chemotaxis activated by LPS and IgG IC primed biological fluids is not related to CXCL2. (A) C57Bl/6 mice were injected with (LPS 10 mg/ kg) i.p. Two hours later mice were sacrificed and PLF sam- ples were evaluated. Measurement of CXCL2 content of PLF by ELISA (left) and PLF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (***P < 0.001, deter- mined by 2-tailed Student´s t test). (B) C57Bl/6 mice were exposed to IgG IC hypersensitivity alveolitis. Four hours later mice were sacri- ficed and BALF samples were evaluated. Mea- surement of CXCL2 content of BALF by ELISA (left) and BALF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 experi- ments (***P < 0.001, determined by 2-tailed Stu- dent´s t test) (C) PMN cells from C57Bl/6 mice were incubated with EGTA and assayed for effi- cient PLF- (left) or BALF-elicited (right) chemo- taxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 indepen- dent experiments, each performed in dupli- cate (*P < 0.05; **P < 0.01, determined by 2- tailed Student´s t test). (D) PMN cells from C57Bl/6 mice were incubated with a Ca2+- specific chelator, EGTA and assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Tran- swell migration assays. Results are expressed as mean ± SEM of n = 4 independent exper- iments, each performed in duplicate. (E) PMN cells from Orai1−/−and matched Orai1+/+ BM chimeras were assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Transwell migra- tion assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (ns = nonsignificant, determined by 2-tailed Student´s t test). (F) Orai1−/−or Orai1+/+ BM chimeras were injected with 200μl of CXCL2 (1 μg/ml) or 200 μl of PBS. 2 hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4 mice per group (ns = non- significant, determined by 2-tailed Student´s t test).

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Chemotaxis Assay, Injection, Enzyme-linked Immunosorbent Assay, Incubation, Migration

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 6. Normal C5a and CXCL2 production, but reduced versus enhanced LPS-induced peritoneal neutrophil accumulation in Orai1−/−and Stim1−/−

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques:

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 7. Orai1 and STIM1 act at distinct levels of the inflammatory cascade in IgG IC-induced hypersensi- tivity pneumonitis. Orai1−/−(A-F) or Stim1−/−(G-L) and matched wild-type chimeras were either exposed to IgG immune complex lung injury. 4 hours later, mice were sacrificed and lung tissue injury and BAL fluid samples were evaluated. (A, G) Functional detection of bioactive C5a in BALF from IC-challenged BM chimeras. Chemotactic activity was determined with Transwell migration assays of neutrophils (PMNs isolated from BM of C57BL/6 and C5aR–/– mice). (B, H) Measurement of CXCL2 content of BALF by ELISA. (C, I) Measurement of TNF-α content of BALF by ELISA. (D, J) Lung H&E section (original magnification 40x). (E, K) Evaluation of lung interstitial PMN infiltration by measurement of MPO activity, normalized to the weight of the lavaged lung. (F, L) Evaluation of PMN accumulation in BALF. Results are expressed as mean ± SEM of n = 5 mice per group (*P < 0.05; **P < 0.01;***P < 0.001, determined by 2-tailed Student´s t test). Groups of 2–3 Orai1−/−or Stim1−/−BM chimeras and 2–3 matched wild-type mice were assayed together.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Functional Assay, Activity Assay, Migration, Isolation, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 8. A summarized model of the distinct roles of STIM1 and Orai1 in IC inflammation and of SOCE vs. nonSOCE in the activation of neutrophils. (A), 1: Initial contact between IC and resident effector cells induces C5a and CXCL2 production that is STIM1-dependent [21]; 2: PMN migration induced by C5a and CXCL2 involves the activation of distinct calcium channels, Orai1 and TRPC6 [30]; 3: TNF-α-mediated regulation of endothelial ICAM-1 promotes PMN adhesion through LFA-1 and Orai1 calcium dynamics [22]; all these steps contribute to the recruitment of PMN to sites of inflammation, but CXCL2 appears not sufficient to overcome C5a dysfunction in the absence of Orai1 (indicated by the dashed arrow). (B) Binding of C5a to the Gαi-coupled C5aR activates Orai1 at the plasma membrane independent from STIM1-mediated SOCE, implicating the existence of a nonSOCE pathway for increased Ca2+ in the regulation of C5aR-mediated PMN migration. The minor role of (IP3-IP3R) Ca2+ store depletion and SOCE pathway for C5a-induced neutrophil chemotaxis is indicated by the dashed arrows.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Activation Assay, Migration, Binding Assay, Clinical Proteomics, Membrane, Chemotaxis Assay

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: Time- and dose-dependent cytotoxicity and apoptosis assays on Glomerular Endothelial Cells (GECs) and Podocytes. GECs. Time-dependent XTT citotoxity assay on GECs ( A ) and podocytes ( B ) incubated with a medium containing CK (20 ng/mL TNF-α, 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein) or vehicle alone at different time-points (12, 18, 24, 36, 48 h) Significant differences in viability were observed on both GECs and podocytes at 18, 24, 36, and 48 h (* p < 0.05, CK vs. vehicle). At 12 h, only the GECs (A) had different viability between treated (CK) and control cells (vehicle, * p < 0.05, CK vs. vehicle). Cytotoxicity tests by XTT ( C ) and apoptosis by TUNEL assay ( D ), evaluating the protective dose of EPC-derived EVs on GECs (black columns) and podocytes (white columns) after 24 h. Incubation with CK alone was effective in inducing cytotoxic (C) and apoptotic damage (D) in both cell types (* p < 0.05, CK vs. vehicle). Adding 1 or 10 μg/mL EPC-derived EVs to cells incubated with CK did not significantly reduce cell damage (* p < 0.05, CK + 1 μg/mL EV and CK + 10 μg/mL EV vs. vehicle), except with GEC incubated with 10 μg/mL EV in TUNEL assay (§ CK + 10 μg/mL EV vs. CK). In contrast, the adding of 25 or 50 μg/mL EPC-derived EVs to cells incubated with CK significantly reduced cell damage at the level of GECs and podocytes (§ p < 0.05, CK + 25 μg/mL EV and CK + 50 μg/mL EV vs. CK). XTT assays are reported as average optical density (O.D.) intensity ± 1SD. For TUNEL assays, data are expressed as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments for each test and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test.

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Incubation, Recombinant, Control, TUNEL Assay, Derivative Assay, Comparison

Journal: Cells

Article Title: Extracellular Vesicles Derived from Endothelial Progenitor Cells Protect Human Glomerular Endothelial Cells and Podocytes from Complement- and Cytokine-Mediated Injury

doi: 10.3390/cells10071675

Figure Lengend Snippet: EPC-derived EVs protected GECs from complement- and cytokine-induced damage ( A , B ). Graphs showing GEC cytotoxicity by XTT assay ( A ) and apoptosis by TUNEL assay ( B ). For XTT assays, data are expressed as average OD intensity ± 1 SD, whereas we expressed TUNEL assays data as the average number of green fluorescent apoptotic cells ± 1 SD. We performed three experiments with similar results for all the assays and the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and Student’s t -test. ( C , D ) FACS analysis ( C ) and representative micrographs ( D ) of ROS expression of GEC (green staining) by confocal microscopy studies (magnification ×400, scale bar 50 μm). Nuclei were counterstained in blue by 2.5 μg/mL Hoechst. We performed three experiments with similar results for all the assays, and we performed the statistical analysis by ANOVA with Newmann–Keuls multiple comparison test and the Kolmogorov–Smirnov test. Incubation with cytokines 20 ng/mL TNF -α, 2.5 ng/mL IL-6, plus 50 ng/mL human recombinant C5a protein CKs significantly increased GEC vitality ( A ), inhibited resistance to apoptosis ( B ), and increased oxidative stress (C) in comparison to treatment with vehicle alone (vehicle, * p < 0.05 CK vs. vehicle). EV stimulation significantly inhibited these effects (§ p < 0.05 CK + EV vs. CK), but not EV were pre-treated with 1 U/mL RNase (# p < 0.05 CK + EV RNase vs. CK + EV).

Article Snippet: In selected experiments, we incubated cells in an appropriate medium containing 20 ng/mL tumor necrosis factor (TNF)-α (Sigma Aldrich), 2.5 ng/mL IL-6, and 50 ng/mL human recombinant C5a protein (R&D Systems, Minneapolis, MN, USA) in the presence or absence of different concentrations of EPC-derived EVs assessed by Nanosight analysis.

Techniques: Derivative Assay, XTT Assay, TUNEL Assay, Comparison, Expressing, Staining, Confocal Microscopy, Incubation, Recombinant