Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 1. (A) Flow cytometric analysis of surface GARP and CD133 on different GSCs and the control, non-stem, GB cell line, T98G. Doublets, debris, and dead cells were excluded from the analysis. Mean fluorescence intensity (MFI) was normalized to the MFI of the respective unstained control. (B) Confocal images of GARP- and nestin-expressing GSCs and T98G. Cells were stained for GARP (red) and nestin (green). Cells were counterstained for their nuclei with Hoechst (blue). Note the intranuclear localization of GARP (NU+). Scale bar corresponds to 20 µm. (C) Percentage of GARPNU+ cells were determined by counting GARP stained nuclei. “No.” indicates the number of counted cells for the analysis.

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Control, Fluorescence, Expressing, Staining

Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 2. GARP expression in isogenic GSCs derived from newly diagnosed GB IT-726. Flow cytometric analysis of IT-726-1, -2, -3a, -3b, and -4. Doublets, debris, and dead cells were excluded from analysis. Mean fluorescence intensity (MFI) was normalized to the MFI of the unstained control. Histograms display one representative result of three independent measurements. Data are displayed as mean values ± SEM.

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Expressing, Derivative Assay, Fluorescence, Control

Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 3. Analysis of expression and localization of GARP in isogenic GSC cell lines, which vary in differentiation states derived from different regions of the same tumor. (A) Number of GARP positive nuclei for GSC lines IT-726—1, -2, -3A, and -4 were analyzed by counting double positive (Hoechst and GARP) cell nuclei (NU+). “No.” indicates the number of counted cells for the analysis. (B) Confocal images of GARP- and nestin-expressing GSC IT-726 -2 and -4. Cells were stained for their nuclei with Hoechst (blue), GARP (red), and nestin (green). Note the intranuclear localization of GARP. Scale bar corresponds to 20 µm.

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Expressing, Derivative Assay, Staining

Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 4. Quantitative assessments of self-renewal capacity by extreme limiting dilution assay (ELDA). (A) Representative results. (B) The pooled results from three independent experiments are indicated in the table. GARPhigh and GARPlow correspond to isogenic GSCs differing in their GARP expression, which were FACs sorted from the GSC line #1095. Estimates of the stem cell frequency (SCF) are framed in red, while lower and upper indicate the confidence intervals for 1/SCF. Statistical significance between groups was calculated by chi-squared tests.

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Limiting Dilution Assay, Expressing

Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 6. GARP expression in GB is unaffected throughout therapy. (A) Survival analysis of GARP and CD133 based on data available through The Cancer Genome Atlas (TCGA). GARP and CD133 mRNA expression data of 152 primary glioblastomas were divided 50/50 into either “low” expres- sion or “high” expression and were analyzed for patient survival. (B) Retrospective analysis of transcriptomic data of 155 GB samples from 28 patients of Kim et al., 2020 [32]. ndGBs, first, and second recurrent tumors were analyzed for their GARP and CD133 mRNA levels across tumor stages. (C) Flow cytometric analysis of IT-619 and IT-654. Doublets, debris, and dead cells were excluded from analysis. Recurrent IT-654 GSCs exhibited stable surface GARP levels after TMZ and radiotherapy, whereas expression of CD133 decreased after treatment. The MFIs were normalized to the unstained control. n = 3. Significance was calculated by Student´s t-test and is indicated as follows: * p < 0.05, *** p < 0.001, and ns (not significant). (D) Number of GARP-positive nuclei for GSC IT-619 and IT-654 were analyzed by counting double-positive (Hoechst and GARP) cell nuclei. “No.” indicates the number of counted cells for the analysis.

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Expressing, Control

Journal: Cancers

Article Title: Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

doi: 10.3390/cancers15245711

Figure Lengend Snippet: Figure 7. Nuclear GARP is a potential new prognostic biomarker for GB patient survival. (A) Im- munohistochemistry of GARP in glioblastoma. GB (WHO grade IV) with low frequency of labeled

Article Snippet: Antibody specificity was demonstrated using GARP-overexpressing Mewo cells, resulting from transient transfection using the LOX-IMVI Cell Avalanche Transfection Reagent (EZ Biosystems, EZT-LOXI-1, College Park, MD, USA) as well as a LRRC32 overexpression plasmid (Origene, SC116699) and an empty vector control plasmid (Origene, PS100001) (Figure S3).

Techniques: Biomarker Discovery, Labeling

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 1. Glycoprotein A repetitions predominant (GARP) is expressed on the surface of platelets and exists as a soluble factor found in liquid platelet rich fibrin (iPRF). (A,B) iPRF was stained directly for the platelet marker, CD41a, and GARP and underwent flow cytometric analysis. Isotype antibodies were used as a control. (A) Dot blots show one representative result. (B) Bar diagrams of GARP expression show the pooled data of percentages (%) of GARP positive platelets in iPRF and the mean fluorescence intensity (MFI) of GARP (n = 3 ± SD, analyzed by paired Students T-tests). (C) Expression of GARP in iPRF was analyzed via Western blot. iPRF lysate was loaded in increasing amounts. Recombinant soluble GARP (sGARP) was loaded at 1 ng/lane and served as a positive control, whereas β-actin acted as the loading control. (D) Bar graph shows the soluble GARP (sGARP) levels in peripheral blood and iPRF analyzed via ELISA (n = 3 ± SD, ns determined by paired Students T-tests).

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Staining, Marker, Control, Expressing, Western Blot, Recombinant, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 2. Isolated platelets from liquid platelet rich fibrin (iPRF) induce a regulatory phenotype in CD4+ T cells. CD4+ T cells and platelets (Plt.) were cocultured at a ratio of 1 to 50 and were treated with 10 µg/mL anti-GARP antibody (Ab). CD4+ T cells were prelabled with carboxyfluorescein succinimidyl ester (CFSE) to track their proliferation and stimulated with 0.5 µg/mL anti-CD3 and 1.0 µg/mL anti-CD28 Abs in the presence or absence of platelets. (A–C) The expression of

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Isolation, Expressing

Journal: Biomedicines

Article Title: GARP Regulates the Immune Capacity of a Human Autologous Platelet Concentrate.

doi: 10.3390/biomedicines10123136

Figure Lengend Snippet: Figure 4. Liquid platelet rich fibrin (iPRF) polarizes M0-macrophages in a more “M0/M2-like” phenotype through a GARP independent mechanism. Human monocyte derived macrophages were polarized, treated with 400 µL iPRF/well, 10 µg/mL anti-GARP antibody (Ab) or were left untreated for 2 days as indicated. Surface marker expression was analyzed via flow cytometry. Bar diagram shows the mean fluorescence intensity (MFI) of each surface marker normalized to the respective M0 control. Representative histograms of 3 independent experiments are shown (n = 5 donors, bar means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 determined by two-way ANOVA).

Article Snippet: Labeled CD4+ T cells were plated in 48 well plates at 106 cells/mL in X-Vivo 15 (Lonza, #BE02-060F, Basel, Switzerland) and incubated for 3 days in the presence or absence of platelets derived from iPRF at a ratio of 1:50 as well as 10 μg/mL anti-GARP Ab (Origene) before undergoing flow cytometric analysis.

Techniques: Derivative Assay, Marker, Expressing, Cytometry, Control