Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on iLite ® C5a Assay Ready Cells, displaying commercial mAb and small-molecule C5aR1 inhibitors ( a ) and in-house C5aR1 mAbs ( b ). Data were normalized to a stimulation control (added C5a) and analyzed by nonlinear curve fitting. Data points were collected in duplicates across multiple assay plates; each assay plate was repeated three times. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors within a C5a-driven PMN calcium flux assay. A negative inhibition control (IgG1k isotype mAb) and a positive inhibition control (20 μ m avacopan) were included. After first recording the baseline, either C5a or buffer was added, followed by the addition of either ionomycin or buffer. Data are presented as fold increase based on the initially recorded baseline (mean FITC detector MFI). The experiment was repeated three times with the blood of a different anonymous healthy donor per repeat; data points were obtained in singlets. Data are presented as mean + SD (one-sided) for better visualization.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Calcium Flux Assay, Inhibition, Control

Journal: Journal of Innate Immunity

Article Title: Functional Analysis of a Novel Complement C5a Receptor 1-Blocking Monoclonal Antibody

doi: 10.1159/000535084

Figure Lengend Snippet: Assay of C5aR1 inhibitors on C5a-stimulated PMNs ( a–c ), followed by titration of mAb 18-41-6-based F(ab’) 2 fragments versus equimolar concentrations of avacopan ( d , e ). a–c A negative control for C5aR1 inhibition (IgG1k isotype mAb) and a positive control (75 μ m avacopan) were included. Data points were normalized to a stimulation control sample (T10stim) with added C5a. a A baseline control (without incubation and without stimulation, Tb) and an incubation control (incubation and without stimulation, T10) are depicted. Titration curves of mAb 18-41-6-based F(ab’) 2 fragments and avacopan were analyzed by nonlinear curve fitting. The experiments were repeated three times with the blood of two different anonymous healthy donors ( a–c ) or one different anonymous healthy donor per repeat ( d , e ); data points were acquired in singlets. Data are presented as mean ± SD.

Article Snippet: 40 µL of cell suspension was added to each well, followed by incubation at 37°C and 5% CO 2 for 30 min. C5a (R&D systems, cat. no.: 2037-C5) was added to the plate at a final concentration of 4 ng/mL (corresponding molarity: 481.93 p m ) in sample buffer.

Techniques: Titration, Negative Control, Inhibition, Positive Control, Control, Incubation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: The role of HBc on C5AR1 expression in HBV-related hepatoma cells. (A) The role of HBV on the expression of complement receptors in hepatoma cells at mRNA levels detected by real-time PCR. (B) The expression of C5AR1 protein mediated by HBV was measured by western blot. (C) The expression of C5AR1 protein in HBV-negative adjacent tissues (n=40), HBV-positive adjacent tissues (n=50), HBV-negative tumor tissues (n=50), and HBV-related tumor tissues (n=65) assessed by IHC analysis (×400). (D) The role of different viral genes on C5AR1 mRNA expression in hepatoma cells that measured by real-time PCR. (E) The role of different viral genes on C5AR1 protein expression in hepatoma cells. (F) The expression of C5 AR1 mRNA in HBc stably-transfected HCC cells, which transfected with Flag-HBc plasmid and selected by G418. (G) The expression of C5AR1 protein in HBc-positive hepatoma cells. (H) The correlation of C5AR1 protein and HBc protein in HBV-related tumor tissues assessed by IHC analysis, the tumor tissues with low HBc expression (n=24), the tumor tissues with high HBc expression (n=41) (×400). C5AR1, C5α receptor 1; HBc, the cells transfected with HBc plasmids; HBV, the cells transfected with hepatitis B virus (HBV) plasmids; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; Mock, the cells transfected with control plasmids; PCR, polymerase chain reaction. *p < 0.05.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Immunohistochemistry, Polymerase Chain Reaction

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: The effect of the NF-κB pathway in activating the promoter of the C5AR1 mediated by HBc in hepatoma cells. (A) The identification of the regulatory region of C5AR1 promoter mediated by HBc via luciferase reporter gene assay. (B) The information of potential NF-κB and Sp1 binding site in C5AR1 promoter in PGL3-P (−233/+100) plasmid. (C) The activation of p65 in HBc-positive hepatoma cells and control cells. (D) The expression of Sp1 in HBc-positive hepatoma cells and control cells. (E) The effect of inhibition of p65 via BAY11-7082 on C5AR1 protein expression in hepatoma cells. (F) The effect of inhibition of Sp1 via Mithramycin A on C5AR1 protein expression in hepatoma cells. (G) The location of p65 in HBc-positive hepatoma cells and control cells. (H) The effect of inhibition of NF-κB via BAY11-7082 on the activity of C5AR1 promoter in PGL3-P (−233/+100) plasmid in HBc-positive hepatoma cells. (I) The information on the mutations in the NF-κB binding site in PGL3-P (−233/+100) plasmid. (J) The influence of the mutation in the NF-κB binding site on activation of C5AR1 promoter in PGL3-P (−233/+100) plasmid. (K) The inhibition of p65 using shRNA plasmids on the expression of p65 protein in HBc-positive hepatoma cells. (L) The effect of inhibition of p65 via shRNA on the activity of C5AR1 promoter in PGL3-P (−233/+100) plasmid in HBc-positive hepatoma cells. (M) The effect of inhibition of p65 by shRNA on C5AR1 protein expression in hepatoma cells. C5AR1, C5α receptor 1; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; Mock, the cells transfected with control plasmid; NF-κB, nuclear factor κB. *p < 0.05.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Luciferase, Reporter Gene Assay, Binding Assay, Plasmid Preparation, Activation Assay, Expressing, Inhibition, Activity Assay, Mutagenesis, shRNA, Transfection

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: The role of C5AR1 in intracellular signal pathways and inflammatory cytokines in HBc-positive hepatoma cells. (A) The inhibition of C5AR1 mediated by shRNA in the expression of C5AR1 protein in HBc-positive hepatoma cells. (B) The effect of inhibition of C5AR1 expression mediated by shRNA on the activation of JNK, ERK, p38, and P13K (AKT) pathway in hepatoma cells stably-transfected with HBc. (C) The effect of inhibition of C5AR1 expression mediated by shRNA on the expression of TNF-α, IL-6, and IL-1β in HBc-positive hepatoma cells. (D) The effect of inhibition of C5AR1 expression mediated by shRNA on the secretion of IL-6 in HBc-positive hepatoma cells. C5AR1, C5α receptor 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; IL, interleukin; Mock, cells transfected with control plasmid; PI3K, phosphoinositide 3-kinase; TNF-α, tumor necrosis factor α. *p < 0.05.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Inhibition, shRNA, Expressing, Activation Assay, Stable Transfection, Transfection, Plasmid Preparation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: The role of C5AR1 in the growth and migration of HCC cells mediated by HBc. (A) The effect of inhibition of C5AR1 expression by shRNA on the growth of HBc-positive hepatoma cells detected by CCK-8 assay. (B) The effect of inhibition of C5AR1 expression via shRNA on the growth of HBc-positive hepatoma cells assessed by plate clone formation assay. (C) The role of C5AR1 in the growth of hepatoma cells mediated by HBc in nude mice. (D) The inhibition of C5AR1 expression via shRNA on the migration of HBc-positive hepatoma cells detected by transwell assay. (E) The effect of inhibition of C5AR1 expression using shRNA on the migration of HBc-positive hepatoma cells was examined by wound healing assay. C5AR1, C5α receptor 1; CCK-8, Cell Counting Kit-8; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, the HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; HCC, hepatocellular carcinoma; Mock, cells transfected with control plasmid. In Fig. 4A, *p < 0.05, the Mock group compared with HBc group; # p < 0.05, the HBc-shNC group compared with HBc-shC5AR1 group.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Migration, Inhibition, Expressing, shRNA, CCK-8 Assay, Tube Formation Assay, Transwell Assay, Wound Healing Assay, Cell Counting, Transfection, Plasmid Preparation

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: C5α gene expression medicated by HBV or HBc, and the role of C5AR1 intracellular signal pathways and inflammatory cytokines in HBc-positive hepatoma cells stimulated by C5α. (A) The serum levels of C5α in HC, CHB patients, HCC patients with HBV infection (HBV-HCC), and HBV-negative HCC patients (non-HBV-HCC) were detected by ELISA. (B) The role of HBV on the expression of C5α at mRNA levels detected by real-time PCR. (C) The role of HBc on the expression of C5α at mRNA levels. (D) The role of C5AR1 in the C5α-mediated activation of intracellular signaling pathways in HBc-positive hepatoma cells (20 ng/mL). (E) The effect of C5AR1 on C5α-induced expression of TNF-α, IL-6, and IL-1β in HBc-positive hepatoma cells that measured by western blot. (F) The effect of C5AR1 on C5α-induced secretion of IL-6 in HBc-positive hepatoma cells detected by ELISA. C5AR1, C5α receptor 1; CHB, chronic hepatitis B; ELISA, enzyme-linked immunosorbent assay; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; HBV, hepatitis B virus; HC, health controls; HCC, hepatocellular carcinoma; IL, interleukin; Mock, the cells transfected with control plasmid; PCR, polymerase chain reaction; TNF, tumor necrosis factor. *p < 0.05.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Activation Assay, Western Blot, Transfection, Plasmid Preparation, shRNA, Polymerase Chain Reaction

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Hepatitis B Virus Core Protein Mediates the Upregulation of C5α Receptor 1 via NF-κB Pathway to Facilitate the Growth and Migration of Hepatoma Cells

doi: 10.4143/crt.2020.397

Figure Lengend Snippet: The role of C5AR1 in the growth and migration of HBc-positive hepatoma cells stimulated by C5α. (A) CCK-8 assay was used to detect the effect of inhibition of C5AR1 using shRNA on the growth of HBc-positive hepatoma cells stimulated by C5α. (B) Plate clone formation assay was utilized to explore the effect of inhibition of C5AR1 via shRNA on the growth of HBc-positive hepatoma cells stimulated by C5α. (C) Transwell assay was used to assess the inhibition of C5AR1 via shRNA on the migration of HBc-positive hepatoma cells stimulated by C5α. (D) Wound healing assay was utilized to examine the effect of inhibition of C5AR1 by shRNA on the migration of HBc-positive hepatoma cells stimulated by C5α. C5AR1, C5α receptor 1; CCK-8, Cell Counting Kit-8; HBc, cells transfected with HBc plasmid; HBc-shC5AR1, HBc-positive hepatoma cells transfected with shRNA plasmid targeting C5AR1; HBc-shNC, HBc-positive hepatoma cells transfected with shRNA control plasmid; shRNA, short hairpin RNA.

Article Snippet: Recombinant human C5α was obtained from Sino Biological (Bejing, China).

Techniques: Migration, CCK-8 Assay, Inhibition, shRNA, Tube Formation Assay, Transwell Assay, Wound Healing Assay, Cell Counting, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial adhesion to the RTEC. RTEC were pre-treated with C5a (as indicated or otherwise 10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to assessment of bacteria binding to RTEC. (A,B) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (C,D) Bacterial adhesion to RTEC, evaluated by fluorescence microscopy analysis. (C) Representative fluorescence images of tri-iodothyronine and tetramethylrhodamine-labeled J96 adhesion to RTEC, J96 (red), and 4’,6-diamidino-2-phenylindole (blue). Scale bars, 50 µm. (D) Quantification of bacteria adhesion to RTEC corresponding to the images in (C) . Results were expressed as number of bacteria per 10 3 RTEC. (A,B,D) data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test [ (A) n = 12 individual wells per group, (B) n = 8 individual wells per group, (C) n = 6 individual images (×200 magnification) from two coverslips per group]. All results shown are representative of three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial internalization into the RTEC. (A) Bacterial uptake by RTEC, evaluated by bacterial plate count assay. RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to the assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test ( n = 8 individual wells) and representative of three independent experiments. (B,C) Bacterial uptake by RTEC, evaluated by fluorescence microscopy analysis. RTEC were pre-treated with or without C5a (10 nM) for 24 h and subjected to the assay. (B) Representative fluorescence images of (tri-iodothyronine and tetramethylrhodamine-labeled) bacteria internalization into RTEC. Bacteria (red), F-actin (green), and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. Lower panel image corresponding to the boxed region in the top-right image shows the cross-sectional views in z stack (bottom and side panel) of RTEC, F-actin, and bacteria, demonstrating J96 within the cytoplasm of RTEC. A representative of three experiments is shown. (C) Quantification of bacteria uptake by RTEC corresponding to the images in (B) . Results were expressed as number of bacteria per 10 3 RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 15 individual images per group). * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Fluorescence, Microscopy, Labeling, Bacteria, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Effect of C5a stimulating renal tubular epithelial cell (RTEC) on bacterial transmigration through the monolayer of RTEC. Bacterial transmigration in RTEC that had been pre-treated with or without C5a (10 nM) for 24 h, then incubated with or without bacteria up to 3 h. (A) Transmigrated bacteria recovered from the lower chamber of RTEC cultures, evaluated by plate count assay. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 4 individual wells per group). (B–D) Epithelial barrier damage, evaluated at 2 h after incubation with bacteria by reduction of tight junction marker ZO-1 expression. (B) Western blot analysis for ZO-1 in RTEC. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 3 individual images per group). (C) Representative fluorescence images of ZO-1 (green) staining in RTEC. Scale bars, 10 µm. (D) Quantification of ZO-1-positive cells, shown as C5a treatment relative to control, corresponding to the RTEC shown in (C) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 4 individual images (×200 magnification) per group]. * P < 0.05, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Transmigration Assay, Incubation, Bacteria, Marker, Expressing, Western Blot, Two Tailed Test, Fluorescence, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: C5a stimulation upregulates Man expression in renal tubular epithelial cell (RTEC). RTEC were pre-treated with C5a (10 nM) and/or LPS (800 ng/mL) or with C5a, in the presence of C5aR1 antagonist (PMX53, 5 µM) or vehicle (Ctrl) for 24 h and subjected to assessment of Man expression and bacteria binding to RTEC. (A,B) Fluorescence microscopy analysis. (A) Representative fluorescence images of Man expression in RTEC (non-permeabilized). Man (green) detected by fluorescein-labeled Galanthus nivalis lectin (GNL) and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (B) Quantification of Man expression, shown as relative fluorescence intensity in GNL-positively stained area corresponding to the images shown in (A) . (C,D) Fluorescence intensity-based microplate assay. (C) Man expression in RTEC that had been treated with C5a and/or LPS. (D) Man expression in RTEC that had been treated with C5a and PMX53. (E) Bacterial adhesion to RTEC, evaluated by bacterial plate count assay. (B–D) Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [ (B) n = 6 coverslips per group, under ×100 magnification, (C) n = 9 individual wells per group, (D,E) n = 6–8 individual wells per group]. All results shown are representative of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: Ctrl, control; RFI, relative fluorescence intensity; RFU, relative fluorescence units.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Expressing, Bacteria, Binding Assay, Fluorescence, Microscopy, Labeling, Staining, Control

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Uropathogenic Escherichia coli adhesion to renal tubular epithelial cell (RTEC) through binding to Man on the cell surface of the RTEC. (A) Confocal microscopic images of bound bacteria in RTEC (non-permeabilized) that had been incubated with labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin and 4’,6-diamidino-2-phenylindole (blue) are shown. Left image: compressed image. Scale bars, 10 µm. Right image: corresponding to the boxed region in the left image show the cross-sectional views in z stack (bottom and side panel) of RTEC, Man, and bacteria, demonstrating association of Man and J96 at cell surface of RTEC. (B) Bacteria binding to RTEC, evacuated by bacterial plate count assay. RTEC were incubated with d -mannose or glucose (1 or 5%) for 30 min before the addition of bacteria. Data were analyzed by two-way ANOVA with multiple comparisons ( n = 8 individual wells per group). (C,E) Man expression in RTCE that had been pre-treated without C5a (C) or with C5a (E) for 24 h and then treated with buffer alone (PBS) or containing α-mannosidase (5 mM) or control enzyme (β-galactosidase) for 1 h, evacuated by fluorescence intensity-based microplate assay. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 6 individual wells per group). (D,F) Bacterial binding to RTEC that had undergone the same treatments as described in (C,E) , evacuated by bacterial plate count assay. (D) Without C5a pre-treatment. (F) With C5a pre-treatment. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 8 individual wells per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. All results shown are representative of three independent experiments.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Binding Assay, Bacteria, Incubation, Labeling, Expressing, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Intracellular signaling/molecules responsible for the action of C5a on Mannosyl residue expression. (A,B) Western blot analysis for ERK1/2 and IκB phosphorylation in renal tubular epithelial cell (RTEC) after C5a (10 nM) stimulation for up to 60 min. In each set of blots, the top row of bands corresponds to incubating membrane with appropriate anti-phospho-antibody and the bottom row of bands corresponds to incubating membrane with appropriate total antibody. Relative amounts of protein phosphorylation are shown in the lower panel of each set of blots. Data were analyzed by one-way ANOVA ( n = 3/group, resulting from three independent experiments). (C) Effect of inhibition of ERK1/2 pathway on Man expression in RTECs assessed by fluorescence intensity-based microplate assay. RTEC monolayers were pre-incubated with C5a for 24 h in the presence of ERK1/2 pathway inhibitor (U0126, 10 μM) and the vehicle control (DMSO) then were used for quantification of Man expression. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9–12 individual wells per group). A representative result of three experiments is shown. (D,E) Production of TNF-α in RTEC that had been treated with C5a (10 nM) and/or LPS (800 ng/mL) for 24 h and subjected to RT-qPCR (D) and ELISA (E) . Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons ( n = 3/group, resulting from three independent experiments). (F) Man expression in RTEC that had been pre-treated with recombinant human TNF-α (10 ng/mL) or vehicle control (BSA) for 24 h, evaluated by fluorescence intensity-based microplate assay. Data were analyzed by unpaired two-tailed Student’s t -test ( n = 9 replicate wells/group). A representative result of three experiments is shown. (G,H) Bacterial adhesion to RTEC. (G) Representative microscopic images of bacteria adhesion to RTEC that had been pre-treated with TNF-α for 24 h, then incubated with tri-iodothyronine and tetramethylrhodamine-labeled J96 for 1 h. Bacteria (red), Man (green) detected by fluorescein-labeled Galanthus nivalis lectin, and 4’,6-diamidino-2-phenylindole (blue) are shown. Scale bars, 25 µm. (H) Quantification of bound bacteria corresponding to the images shown in (G) . Data were analyzed by unpaired two-tailed Student’s t -test [ n = 6 individual images (×200 magnification) from two coverslips per group]. A representative result of three experiments is shown. * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Residue, Expressing, Western Blot, Phospho-proteomics, Membrane, Inhibition, Fluorescence, Incubation, Control, Two Tailed Test, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Recombinant, Bacteria, Labeling

Journal: Frontiers in Immunology

Article Title: Epithelial C5aR1 Signaling Enhances Uropathogenic Escherichia coli Adhesion to Human Renal Tubular Epithelial Cells

doi: 10.3389/fimmu.2018.00949

Figure Lengend Snippet: Detection of C5a and C5aR1 in human urinary tract. (A–C) C5a and creatinine concentrations in urine from urinary tract infection (UTI) patients and healthy controls. (A) Urinary C5a concentrations. (B) Urinary creatinine concentrations. (C) Ratios of urinary C5a/creatinine concentration. Data were analyzed by Mann–Whitney U two-tailed test. ** P < 0.01, n.s. no significant. (D) C5aR1 expression in human kidney tissue determined by immunochemical staining. Control: negative control, tissue stained with non-specific mouse IgG2a. Scale bar, 50 µm.

Article Snippet: Co., USA); human recombinant C5a (with an endotoxin level of ≤0.1 EU per 1 μg of protein) (R&D systems).

Techniques: Infection, Concentration Assay, MANN-WHITNEY, Two Tailed Test, Expressing, Staining, Control, Negative Control