Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: (A, B) TEM immunogold localization of sCLU in RBCs membrane protrusions (A) and vesicles (ves) (B) collected from fresh units of stored RBCs (N = 2, young healthy donors). Solid or dashed arrows indicate sCLU immunogold localization at the periphery or the cytosol of the vesicles, respectively. (C) Representative immunoblot analysis of RBCs-derived purified vesicles (N = 2) probed with either polyclonal anti-sCLU or with monoclonal anti-Band 3 antibodies. Molecular weight markers are indicated to the right of the blot. Bars in (A), (B), 100 nm.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Membrane, Western Blot, Derivative Assay, Purification, Molecular Weight

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with polyclonal antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) Anti-dinitrophenylhydrazone (DNP) immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Purification, Immunoprecipitation, Control, Clinical Proteomics, Membrane, Staining, Western Blot, Modification

Journal: PLoS ONE

Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

doi: 10.1371/journal.pone.0026033

Figure Lengend Snippet: Erythrocytic sCLU localizes at both sides of the plasma membrane in association with non-cytoskeletal areas, as well as in the cytosol (see also, Antonelou et al., accompanying paper). At the intracellular side of the RBCs membrane sCLU may bind Band 3, Hb and/or other cytoskeleton-free membrane portions. On the other hand, the sCLU that localizes at the extracellular side of the RBCs membrane can attach to membrane by binding to Band 3, CD59, plasma membrane IgGs or to an currently unknown sCLU-specific receptor. Physiological in vivo or ex vivo RBCs senescence (1) is associated with cytosol, cytoskeleton and membrane structural alterations, including Band 3 modifications, increased membrane binding of IgGs, proteolysis, protein aggregation and increased oxidation defects. Vesiculation (2), a self-protective mechanism of mammalian erythrocytes, removes oxidized proteins and aggregates from both plasma membrane and cytosol thereby postponing the untimely elimination of otherwise healthy erythrocytes. This process takes place through the entire in vivo or ex vivo lifespan of RBCs and is functionally connected to the release of sCLU-, Band 3-, CD59-, Hb- and IgGs-containing vesicles. We propose that vesicular sCLU by following its membrane linkers (e.g. Band 3) or other unknown cytosolic interacting proteins assists via its chaperone function in the disposal of non-functional or death signalling effective material from RBCs.

Article Snippet: The polyclonal antibody against CD59 was obtained from R&D Systems (AF 1987).

Techniques: Clinical Proteomics, Membrane, Binding Assay, In Vivo, Ex Vivo, Functional Assay

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 1. p53 binds to the putative binding sites within the CD59 gene in vitro. A, CD59.1, the first putative p53-responsive element, is located in the 5V flanking region of the CD59 gene ranging from nucleotide 1665 to 1637 upstream of exon 1. CD59.2 is the second putative p53-binding sequence located in intron 1 of CD59 ranging from nucleotide 675 to 645 upstream of exon 2. Possible p53 half-binding sites are marked in capital letters. The differences between these and the p53 consensus binding sequence are underlined. B, EMSA with samples labelled with horseradish peroxidase, incubated with 100 ng wild-type recombinant p53 in the presence of 0.1 Ag/AL poly(deoxyinosinic-deoxycytidylic acid). Oligonucleotides were separated in a 2% agarose gel and detected by autoradiography.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Binding Assay, In Vitro, Sequencing, Incubation, Recombinant, Agarose Gel Electrophoresis, Autoradiography

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 2. p53-dependent expression of CD59 in HeLa cells. A, Western blot analysis of p53 (anti-p53, cloneBP53-12 recognizes all p53 modifications) and CD59 expression in HeLa cells and cells 36 hours after transfection with siRNA specifically blocking expression of p53. Detection of p42 MAPK was done as a control for the siRNA specificity. B, fluorescence-activated cell sorting analysis of CD59 expression in HeLa and p53 siRNA–transfected cells.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Transfection, Blocking Assay, Control, Fluorescence, FACS

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 3. Alterations in CD59 expression in IMR32 cells treated with camptothecin. A, Western blot analysis of CD59 expression in IMR32 cells treated with camptothecin for either 24 or 48 hours. B, expression of CD59 in IMR32 cells treated with camptothecin for 24 or 48 hours detected by quantitative PCR. Expression in untreated cells is set as 100%. Columns, mean for two independent experiments; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison. C, resistance of IMR32 (x) and surviving IMR32 (n) cells to C lysis. Lysis assay with preincubation of both untreated (.) and surviving (E) IMR32 cells with CD59-blocking antibody BRIC229 was carried out as a control. Points, mean for three independent experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Comparison, Lysis, Blocking Assay, Control

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 4. Expression of p53 and recruitment to the CD59 gene. A, two different antibodies were used for detection of p53: anti-p53, clone BP53-12 recognizing all forms of p53 and anti–acetyl-p53 (Lys373, Lys382) to detect the changes in p53 acetylation. B, IMR32 cells were treated with camptothecin for either 24 or 48 hours. Immunoprecipitation was done with anti–acetyl-p53 (Lys373, Lys382) antibody. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (striped columns) in control IMR32 cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Immunoprecipitation, Binding Assay, Control, Chromatin Immunoprecipitation, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 6. Chromatin immunoprecipitation analysis of recruitment of p53 to the CD59 gene in living Hep3B cells. Cells were treated with IFN-g or IL-8 for 36 hours. Immunoprecipitation was carried out either with anti–acetyl-p53 (Lys373, Lys382; A) or anti-p53, clone BP53-12 (B) antibodies. Binding of p53 to its responsive elements CD59.1 (black columns) and CD59.2 (white columns) in untreated cells is set as 1. Columns, mean for two independent chromatin immunoprecipitation experiments each analyzed in duplicate; bars, SD. Compared sets are shown by columns with interrelated Ps for comparison.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Binding Assay, Comparison

Journal: Cancer research

Article Title: p53 regulates cellular resistance to complement lysis through enhanced expression of CD59.

doi: 10.1158/0008-5472.CAN-05-3191

Figure Lengend Snippet: Figure 5. Effect of cytokines on p53 and CD59 expression in IMR32 and Hep3B cells. A, quantitative PCR analysis of p53 (black columns) and CD59 (white columns) expression following an incubation of Hep3B and HL60 cells with IFN-g or IL-8. Results from two independent measurements displayed a significant difference between untreated and cytokine treated Hep3B samples. *, P < 0.05; **, P < 0.01. However, no significant difference was observed between HL60 samples. B, flow cytometry analysis of p53 (black columns; Lys373, Lys382) acetylated p53 (white columns) and CD59 (striped columns) following the same treatments as in (A). *, P < 0.01; **, P < 0.001. Columns, mean of four measurements obtained from two separate experiments; bars, SD.

Article Snippet: Primary antibodies used were mouse monoclonal BRIC229 (International Blood Group Reference Laboratory, Bristol, United Kingdom) for detection of CD59; rabbit polyclonal anti-acetyl-p53(Lys373, Lys382) and mouse monoclonal anti-p53, clone BP53-12 (Upstate, Dundee, United Kingdom) for detection of acetylated and total p53, respectively; rabbit polyclonal antip42 mitogen-activated protein kinase (MAPK; Cell Signaling Technology) for p42 MAPK detection.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Flow Cytometry

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Role of CD59 and expression in different cancers. ( A ) involvement of CD59 in different biological pathways using ShinyGO v0.741. ( B ) Comparison of CD59 mRNA expression between cancer and its normal tissue counterpart in multiple cancers using the TCGA database and the GAPIA2 analytical tool. ( C ) mRNA expression of CD59 in KIRC, CESC, GBM, HNSC, and STAD cancer patients and normal tissue from the TCGA database using the GAPIA2 analytical tool. ( D i – v ) Protein expression of CD59 in KIRC ( i ), CESC ( ii ), GBM ( iii ), HNSC ( iv ), and STAD ( v ) cancer patients and normal tissue from the Human Protein Atlas. Each dot represents mRNA expression of sample and * indicates p ≤ 0.05.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Comparison

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Biological pathways related to the proteins involved in CD59 networks based on KEGG pathways (based on STRING database).

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Coagulation, Infection

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: List of cancers and CD59 expression with significance.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Expression of CD59 in cancer cell lines and cancer patients. ( A ) mRNA expression of CD59 in HEK 293T (normal human embryonic kidney), 786-O (KIRC), HeLa (CESC), and SF188 (GBM) by RT-PCR. ( B – D ) protein expression of CD59 in HEK 293T (normal human embryonic kidney), 786-O (KIRC), HeLa (CESC), and SF188 (GBM) by Western blot ( B ), flowcytometry (surface staining) ( Ci ), and its quantification ( Cii ), and immunofluorescence (surface staining) ( Di ) with its quantification ( Dii ). β-tubulin (control) for B (786-O, HeLa, and SF188) are similar to Figure 7A(iii). Data represents the minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Immunofluorescence, Control

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Prognostic analysis of CD59 in cancer. ( A i – v ) Analysis of CD59 expression and overall survival (OS) using Kaplan–Meier in KIRC ( i ), CESC ( ii ), GBM ( iii ), HNSC ( iv ), and STAD ( v ) using the GEPIA 2 dataset. ( B ) Analysis of clinical relevance of CD59 expression across various cancer types using Timer 2.0 analytical tool.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Correlation between CD59 expression and Treg and MDSC. ( A ) Immune subtype analysis of ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD using TISIDB. ( B ) expression and distribution of CD59 on immune cells by the HPA dataset ( Bi ) and Schmiedel database ( Bii ). ( C ) Flow cytometry analysis (Ci) with quantification (Cii) of FOXP3 intracellular expression on 786-O, HeLa, and SF188 cell lines. Correlation between CD59 and Treg cells ( D ) and MDSC cells ( E ) in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD using TISIDB. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Flow Cytometry

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Correlation between CD59 expression and TAM and immune-infiltrating M2 macrophage: ( A ) Spearman correlation between CD59 and macrophage in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD. ( B ) Heatmap of correlation between CD59 and ( i ) macrophage, IL6, IL6R, and ( ii ) IL10, and IL10RB in KIRC, CESC, GBM, HNSC, and STAD. ( C ) Scattered plot of relationship between M2 macrophage infiltration and CD59 expression in ( i ) KIRC, and ( ii ) GBM, and STAD using Timer 2.0 analytical tool.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Co-culture of tumor cells with macrophage increases infiltrating M2 macrophage: ( A ) Analysis of tumor cells in the co-culture system. ( Ai ) Basic expression of phosphorylated STAT3 (Y705) in tumor cells. ( Aii ) Expression of pSTAT3 (Y705) in tumor cells in the presence or absence of macrophage. ( Aiii ) mRNA expression of IL10 in tumor cells in the presence or absence of macrophage. ( Aiv ) mRNA expression of CD59 in tumor cells in the presence or absence of macrophage. ( B ) Analysis of macrophage in the co-culture system. ( Bi ) Gating strategy to identify M2 macrophage. ( Bii ) Percentage of M2 macrophage in 786-O, HeLa, and SF188. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Co-Culture Assay, Expressing

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: TGF-β-mediated immune suppression in CESC, GBM, HNSC, and STAD. ( A ) Expression analysis of TGFβ1. ( Ai , ii ) Flow cytometry analysis of TGF-β expression on 786-O, HeLa, and SF188 cell line. ( Aiii ) Immunoblotting of TGFβ1 and ( Aiv ) ELISA of secreted TGFβ1. ( B ) Spearman correlation analysis of TGF-β and CD59 expression in ( i ) KIRC, ( ii ) CESC, GBM, HNSC, and STAD and supported by ( Ci ) heat map of TGF-β and CD59 expression in KIRC, CESC, GBM, HNSC, and STAD using TISIDB. ( Cii ) Correlation between CD4+ and CD8+ T cell immune infiltration and CD59 expression in multiple cancers using Timer 2.0 analytical tool. β-tubulin (control) for A(iii) (786-O, HeLa, and SF188) are similar to B. Data represents a minimum of three independent experiments, where * indicates p ≤ 0.05, ** indicates p ≤ 0.01, **** indicates p ≤ 0.0001, t -test.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques: Expressing, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance

doi: 10.3390/cancers16213699

Figure Lengend Snippet: Diagram of CD59-mediated immune suppression in CESC, GBM, HNSC, and STAD. CESC, GBM, HNSC, and STAD have high amounts of FOXP3, IL10, TGFβ1, and pSTAT3, leading to increased CD59 transcription and the recruitment of immune suppressive cells such as MDSC, Treg, and TAM in the TME.

Article Snippet: CD59 (Thermo Fisher Scientific# PA5-78993) primary antibody was diluted in 1% BSA in PBS (antibody dilution buffer).

Techniques:

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Oligonucleotides used for qPCR analysis.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Sequencing, Amplification

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Antibodies and dyes used.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Concentration Assay

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] compared to various controls. A one-day stimulation protocol was used. Ctrl = non-stimulated group, [PEMF(OFF)] = apparatus turned off, sham = apparatus turned on without electromagnetic impulse. n = 5 independent experiments. Mean with standard deviation (SD). Non-stimulated group as control has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Standard Deviation, Control

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] with and without 24 h TNFα pre-stimulation (10 ng/mL) compared to their respective control (ctrl). A one-day stimulation protocol was used. Ctrl = non-stimulation. n = 5 independent experiments. Mean with standard deviation (SD). Non-stimulated group without TNFα pre-stimulation (Ctrl/Ctrl) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons (*). * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Control, Standard Deviation

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (CD55, CD59, CFH and CFI) and cytokines (IL-6 and TNFα) in synovial fibroblast cell line K4IM after PEMF stimulation [PEMF(ON)] for 3 days and 6 days compared to their respective control (ctrl). Ctrl = non-stimulation. n = 4 independent experiments. Mean with standard deviation (SD). Non-stimulated group (3 days) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons (*) * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Gene Expression, Control, Standard Deviation

Journal: Journal of Personalized Medicine

Article Title: In Vitro Investigation of Pulsed Electromagnetic Field Stimulation (PEMF) with MAGCELL ® ARTHRO on the Regulatory Expression of Soluble and Membrane-Bound Complement Factors and Inflammatory Cytokines in Immortalized Synovial Fibroblasts

doi: 10.3390/jpm14070701

Figure Lengend Snippet: ( A ) Representative images of K4IM cell line after 3 days and 6 days PEMF(ON) stimulation compared to their non-stimulated counterparts, respectively (200× magnification), immunolabeled with a CD59 specific antibody and negative control of the staining. Green (Alexa Fluor 488) = CD59, blue (DAPI) = cell nuclei, gray (Phalloidin Alexa Fluor 633) = F-Actin cytoskeleton. Scale bar = 100 µm. ( B ) Graphic representation of relative CD59 protein fluorescence intensity, n = 4 independent experiments, mean with standard deviation (SD). Control (3 days) has been normalized to 100. Repeated measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Mouse anti-human CD59 , Bio-Rad, Bio-Rad, Feldkirchen, Germany , MCA1054 , 1 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: The Journal of Clinical Investigation

Article Title: Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria

doi: 10.1172/JCI74747

Figure Lengend Snippet: The primary event (PNH5) is represented in A and B; the secondary event (PNH1) is represented in C–F. (A) Analysis of VAFs of the mutations identified in case PNH5 indicated that the KDM3B mutation was present at a lower frequency than the PIGA mutation, and both mutations were almost exclusively confined to the sorted PNH+ (CD59–) fraction. (B) Single-colony sequencing results confirmed that the PIGA and KDM3B mutations were present in the same cell population. (C) Deep sequencing VAFs for PIGA-1 (G68E), PIGA-2 (splice site), and NTNG1 (P24S) mutations, all of which were primarily present in the PNH fraction in the PNH1 case. (D) Bacterial subcloning and Sanger sequencing results demonstrated that the PIGA mutations in this case were independent, suggesting the presence of 2 separate PNH clones. (E) Single-colony sequencing further confirmed that 2 independent PNH clones were present and also suggested that the PIGA splice site mutation appeared to be a secondary event preceded by a NTNG1 mutation. (F) The combination of deep sequencing data with single-colony sequencing allowed for a representation of the clonal architecture in PNH1.

Article Snippet: Whole blood was stained with antibodies against GlyA, CD15, CD24, CD55, CD59 (Beckman Coulter), CD59 (Life Technologies), and FLAER (Alexa 488 Proaerolysin Variant; Cedarlane Scientific) in various combinations to quantitate the number of GPI-deficient rbc and wbc in each patient.

Techniques: Mutagenesis, Sequencing, Subcloning, Clone Assay