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

Article Title: Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217.

doi: 10.4049/jimmunol.1502701

Figure Lengend Snippet: FIGURE 2. miR-200 (b and c) regulates cell sensitivity to complement, C5b-9 deposition, and CD46 and CD55 expression. (A) K562 cells were transfected with miR-200b/c or control plasmid. After 3 or 24 h, cells were treated with Ab and NHS. Cell death (%) was measured by trypan blue inclusion. (B) K562 cells were transfected with miRNA inhibitors specific for miR-200b and miR-200c, or with nonspecific oligonucleotide as negative control. After 24 h, cells were treated with Ab and complement, as above. Cell death (%) was measured by propidium iodide inclusion. (C) K562 cells transfected with miR-200b/c or a control plasmid for 24 h were treated with a sublytic dose of Ab and then with NHS for 10 min at 37˚C. Then they were labeled with goat anti-C3 Ab and FITC-conjugated secondary Ab and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values of cell-bound C3b, representative of three independent experiments, are shown. (D) K562 cells transfected with miR-200b/c or a control plasmid for 24 h were treated with sublytic complement, as in (C). Then they were labeled with mouse anti– C5b-9 aE-11 Ab and FITC-conjugated secondary Ab and analyzed by flow cytometry. MFI values of cell-bound C5b-9, representative of three independent experiments, are shown. (E) K562 cells transfected for 24 h with inhibitors of miR-200b and miR-200c, or with nonspecific oligonucleotide, were incubated with sublytic Ab and NHS for 10 min at 37˚C. The cells were labeled with the aE-11 Ab, as above. MFI values of cell-bound C5b-9, representative of three independent experiments, are shown. (F) K562 cells were transfected with miR-200b/c or control plasmid for 24 h and were labeled with mouse anti-CD46, anti-CD55, or anti- CD59 Ab and then with FITC-conjugated secondary Ab. Cells were then analyzed by flow cytometry, and MFI values were determined. Results, representative of three independent experiments, present each regulator’s level relative to its expression in control cells (set as 100%). *p , 0.05, **p , 0.01 relative to control.

Article Snippet: Mouse anti-human CD46, anti-human CD55, and anti-human CD59 mAbs were purchased from AbD Serotec (Oxford, U.K.).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Labeling, Cytometry, Incubation

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

Article Title: Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217.

doi: 10.4049/jimmunol.1502701

Figure Lengend Snippet: FIGURE 3. miR-217 regulates cell sensitivity to complement, C3 and C5b-9 deposition, and CD46 expression. (A) K562 cells were transfected with miR-217 or control plasmid. After 3 or 24 h, cells were treated with Ab and NHS (60 min). Cell death (%) was measured by trypan blue inclusion. (B) K562 cells were transfected with miRNA inhibitors specific for miR-217 or with nonspecific oligonucleotide as negative control. After 24 h, cells were treated with Ab and complement, as above. Cell death (%) was measured by propidium iodide inclusion. (C and D) K562 cells transfected for 24 h with miR-217 or control plasmid were incubated with a sublytic dose of Ab and NHS for 10 min at 37˚C. Cells were labeled with goat anti-C3b Ab and FITC-conjugated secondary Ab (C) or mouse anti–C5b-9 aE-11 Ab and FITC-conjugated secondary Ab (D) and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values of cell-bound C3b or C5b-9, representative of three independent experiments, are shown. (E) K562 cells transfected for 24 h with miR-217 inhibitor or with nonspecific oligonucleotide (C) were incubated with a sublytic dose of Ab and NHS for 10 min at 37˚C. The cells were labeled with aE-11 Ab and FITC-conjugated secondary Ab, and MFI values of cell-bound C5b-9 were determined. Results are representative of three independent experiments. (F) K562 cells were transfected with miR-217 or control plasmid for 24 h and were then labeled with mouse anti-CD46, anti-CD55, or anti-CD59 Ab and FITC-conjugated secondary Ab. Cells were then analyzed by flow cytometry, and MFI values were determined. Results, representative of three independent experiments, present each regulator’s level relative to its expression in control cells (set as 100%). *p , 0.05, **p , 0.01 relative to control.

Article Snippet: Mouse anti-human CD46, anti-human CD55, and anti-human CD59 mAbs were purchased from AbD Serotec (Oxford, U.K.).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Negative Control, Incubation, Labeling, Cytometry

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: NMR structure of the protein backbone of human CD59. The figure shows the 20 lowest energy structures of human CD59 with all lysine side chains and H44 (PDB Id: 1CDQ) (17). The structures were superimposed for the backbone of the β turn 41–44. The square highlights the K41–H44 glycation motif. K41 is within 5.91 ± 1.44 Å of the D1 imidazolic nitrogen of H44.

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques:

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: Glycation abrogates the homologous restriction activity of human CD59. A 10% cell suspension of GPE was incubated with immunoaffinity-purified hRBC CD59 previously exposed for different time intervals to glucose (▵, 0.5 M) or ribose (□, 0.5 M), or to nonglycating sorbitol (■, 0.5 M). The sensitivity of GPE to human MAC was then tested by using purified human C5b6 and C7, C8, and C9. Each point represents the mean of triplicate determinations (SEM smaller than data points). Representative of three experiments with comparable results. (Inset) Number of CD59 molecules incorporated per GPE, determined with 125I-CD59 before (C) and after (R) glycation with ribose.

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques: Activity Assay, Incubation, Purification

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: Glycated CD59 in human urine. (A) Urine samples from diabetic (lanes 2 and 3) and nondiabetic (lanes 1, 4, and 5) subjects were concentrated by ultrafiltration and separated by anion exchange chromatography, and fractions were dot-blotted for the presence of CD59 with anti-CD59-specific Ab. CD59-positive fractions were pooled and immunoprecipitated with the HC1 anti-CD59-specific Ab. Immunoprecipitates were spun down and boiled for 30 min; then the immunocomplexes were separated by SDS/PAGE and immunoblotted with the monoclonal YTH53.1 anti-CD59 Ab (A, Upper), and with the anti-hexitol–lysine Ab (A, Lower). The Upper samples were separated by using a 20-cm-long gel that resolved the multiple CD59 bands (mw = molecular weight markers). The Lower samples were separated by using a minigel. Immunoaffinity-purified hRBC CD59 was included in the Upper gel as a control (lane 6). The signal intensity of the immunoblot bands marked by the arrows was quantified by BIOQUANT image analysis software. HbA1c levels were measured at the clinical laboratory of The Joslin Diabetes Center by HPLC. (B) Immunoblot of glycated (G) and nonglycated (NG) albumin (Upper) and immunoaffinity-purified hRBC CD59 (Lower) with anti-hexitol–lysine Ab.

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques: Chromatography, Immunoprecipitation, SDS Page, Molecular Weight, Purification, Western Blot, Software

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: Site-directed mutagenesis of K41 or H44 abrogates the sensitivity of human CD59 to glycation–inactivation. (A) WT and CD59–Gln-41 or CD59–Gln-44 mutants were expressed in CHO cells. Expression was confirmed by immunocytochemistry by using the anti-human CD59 monoclonal Ab YTH53.1 and fluorescence (Texas red)-labeled anti-rat IgG secondary Ab: ■, transfection vector only; □, WT CD59; ▵, Gln-41 mutant CD59; and ○, Gln-44 mutant CD59. (B) Recombinant WT and mutant CD59 were immunoaffinity purified from CHO cells, and their activity was tested in the GPE hemolytic assay (symbols as in A). (C) Activity of immunoaffinity-purified WT and mutant CD59s before and after glycation with ribose for different time intervals. The points represent the mean of triplicate determinations (SEM smaller than the data points). Representative of three experiments with comparable results.

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques: Mutagenesis, Expressing, Immunocytochemistry, Fluorescence, Labeling, Transfection, Plasmid Preparation, Recombinant, Purification, Activity Assay, Hemolytic Assay

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: Glycation increases the sensitivity of hRBC to MAC-mediated lysis and of HUVEC to MAC-induced release of growth factors. (A) hRBC were incubated without or with 50 mM ribose for 48 h at room temperature followed by reduction with NaBH3CN (open column, cells not exposed to NaBH3CN; filled columns, cells exposed to NaBH3CN). Cell volume was adjusted in glycated and nonglycated control cells, and aliquots of cells were separated for rescue with immunoaffinity-purified human CD59 (4 μg) or incubation with neutralizing anti-CD59 Ab (YTH53.1), before exposure to purified human C5b6, C7, C8, and C9 to form the MAC. The last column to the right represents cells that were first exposed to the anti-CD59 Ab and then rescued with affinity-purified human CD59. (Inset) Similar osmotic fragility of control (□) and glycated (■) cells after volume adjustment. (B) HUVEC were incubated with or without 50 mM ribose (24 h, 37°C), followed by reduction with NaBH3CN. Cells were then exposed to purified human C5b6, C7, C8, and C9 to form the MAC, and the mitogenic activity in the conditioned medium was measured as in ref. 13. Results are expressed as the ratio of mitogenic activity released into the conditioned media in the presence or absence of MAC.

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques: Lysis, Incubation, Purification, Affinity Purification, Activity Assay

Journal:

Article Title: Molecular basis for a link between complement and the vascular complications of diabetes

doi:

Figure Lengend Snippet: Alignment of CD59 amino acid sequences from different species around residue W40

Article Snippet: CD59 from hRBC butanol extracts and CHO lysates was purified by immunoaffinity chromatography as described ( 21 ) by using the monoclonal rat anti-human CD59 Ab YTH53.1 (Serotec).

Techniques:

Journal: Molecular Medicine Reports

Article Title: The effect of anti-HLA class I antibodies on the immunological properties of human glomerular endothelial cells and their modification by mTOR inhibition or GCN2 kinase activation

doi: 10.3892/mmr.2021.11994

Figure Lengend Snippet: Effect of anti-HLAI on ICAM-1, HLA-DR, CD46 and CD59, and the impact of halofuginone or everolimus treatment. (A) Representative experiment for each of the evaluated factors. (B) Cumulative results are presented. Anti-HLAI antibodies upregulated ICAM-1, HLA-DR, CD46 and CD59. Halofuginone or everolimus treatment decreased ICAM-1. Data are presented as the mean ± SEM. *P<0.05 vs. control cells, # P<0.05 vs. anti-HLAI-treated cells, ^ P<0.05 vs. anti-HLAI-treated cells administered halofuginone, + P<0.05 vs. anti-HLAI-treated cells administered everolimus and $ P<0.05 vs. anti-HLAI-treated cells with halofuginone and everolimus. HLAI, human leukocyte antigen class I; ICAM-1, intracellular adhesion molecule-1; Hal, halofuginone; Ever, everolimus; Ctrl, control.

Article Snippet: Blots were incubated at 4°C for 16 h with the primary antibodies specific against activated cleaved caspase-3 (cleaved caspase-3, 1:1,000, cat. no ab13847, Abcam), focal adhesion kinase (FAK, 1:100, cat. no sc-271126, Santa Cruz Biotechnology, Inc.), phosphorylated at Tyr397 FAK (p-FAK, 1:1,000, cat. no 8556, Cell Signaling Technology, Inc.), mTOR (1:100, cat. no sc-517464, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser2448 mTOR (p-mTOR, 1:100, cat. no sc-293133, Santa Cruz Biotechnology, Inc.), p70S6 kinase (p70S6K, 1:100, cat. no sc-8418, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr389 p70S6K (p-p70S6K, 1:1,000, cat. no 9234, Cell Signaling Technology), protein kinase B (Akt, 1:100, cat. no sc-5298, Santa Cruz Biotechnology, Inc.), phosphorylated at Ser474 Akt (p-Akt, 1:1,000, cat. no 4060, Cell Signaling Technology, Inc.), GCN2 kinase (GCN2K, 1:100, cat. no sc-374609, Santa Cruz Biotechnology, Inc.), phosphorylated at Thr899 GCN2K (p-GCN2K, 1:1,000, cat. no ab75836; Abcam), eIF2α (1:100, cat. no sc-133132, Cell Signaling Technology, Inc.), phosphorylated at Ser51 eIF2α (p-eIF2a, 1:1,000, cat. no 9721, Cell Signaling Technology, Inc.), intercellular adhesion molecule 1 (ICAM-1, 1:1,000, cat. no 4915; Cell Signaling Technology), HLA-DR (Ultra-LEAFTM Purified anti-human HLA-DR Antibody, cat. no 307648, Biolegend), CD46 (1:1,000, cat. no CSB-PA923298, Cusabio), CD59 (1:1,000, cat. no CSB-PA004947YA01HU, Cusabio), and β-actin (1:5,000, cat no. 4967, Cell Signaling Technology, Inc.).

Techniques: Control

Journal:

Article Title: Regulated complement deposition on the surface of human endothelial cells: Effect of tobacco smoke and shear stress

doi: 10.1016/j.thromres.2007.11.005

Figure Lengend Snippet: Changes in cell surface expression of complement regulators (Mean ± S.D.), CR1 (CD35) (n = 3), DAF (CD55) (n = 4), and Protectin (CD59) (n = 4) in response to BMEC exposure to TS (0.03 puffs/ml), chemical (LPS at 1 μg/ml or INF-γ at 100 U/ml), or mechanical shear stress (18 dyne/cm2). Results were normalized to baseline CD35, CD59 and Cd59 expression observed on untreated BMEC monolayers. The data were analyzed by single factor ANOVA. (*) Denotes a statistically significant increase (P<0.05).

Article Snippet: Murine monoclonal anti-human CD35, CD55, and CD59 antibodies were obtained from Ancell Corporation (Bayport, MN), and used at concentrations of 1 μg/ml, 10 μg/ml, and 5 μg/ml, respectively.

Techniques: Expressing

Journal: Journal of Oral Microbiology

Article Title: Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76

doi: 10.1080/20002297.2022.2105013

Figure Lengend Snippet: Mode of receptor recognition of DLY and other CDCs. Each recombinant CDC (rDLY, rSm-hPAF, rILY, and rSLY) was incubated with human erythrocytes in the presence or absence of cholesterol and/or human CD59 monoclonal antibody (YTH53.1). Triplicate samples were assayed at least twice each. Representative results are shown as hemolysis averages with standard deviations (SD). Significance of differences between hemolysis in the absence (dark-gray bar) and presence of inhibitor(s) for receptor binding of CDCs (cholesterol only, cyan bar), YTH53.1 only (magenta bar), and both (purple bar) was evaluated using F-tests followed by Welch’s t-tests or Student t-tests (**p < 0.01, *p < 0.05).

Article Snippet: To inhibit the binding between recombinant CDC and human CD59 on erythrocytes, we pre-incubated anti-human CD59 monoclonal antibody (YTH53.1; DS Pharma Biomedical Co., Ltd., Suita, Osaka, Japan) with human erythrocytes at a final concentration of 50 μg/mL for 15 min at 37°C and then washed them with PBS.

Techniques: Recombinant, Incubation, Binding Assay