Journal: The American Journal of Pathology

Article Title: Aberrant Expression of ID2, a Suppressor of B-Cell-Specific Gene Expression, in Hodgkin's Lymphoma

doi: 10.2353/ajpath.2006.060020

Figure Lengend Snippet: Quantitative real-time PCR and Western blot analysis of ID2, E2A, PAX5, CD19, CD79A, and BLNK expression in HRS-cell and other B-cell (lymphoma) cell lines. For all PCR, predesigned assays from ABI were used (Assays-on-demand). ΔCt values relative to B2M are shown as fold expression relative to B2M. For the PAX5 and CD79A ΔCts, to all values 1 and 3, respectively, were added to obtain positive numbers. A: Expression of ID2, E2A, and PAX5 in four cell lines derived from classical HL (L1236, L428, KMH2, and HDLM2), one cell line derived from lymphocyte-predominant HL (DEV), one Epstein-Barr virus-transformed B-cell line (NCNC), two MCL cell lines (NCEB1 and Granta 519), two DLBCL cell lines (SuDHL6 and OCI-LY7), and two BL cell lines (Raji and DG75) relative to B2M are shown. Bars represent average values. B and C: ID2 expression relative to PAX5 and E2A is shown. D: Expression values for the B-cell markers CD19, CD79A, and BLNK relative to B2M are shown. E: ID2, E2A, and PAX5 protein expression analyzed by Western blotting in the cell lines indicated is shown. Loading of equal amounts of protein was confirmed using an anti-actin antibody.

Article Snippet: One-microliter aliquots of the cDNAs were used as templates in real-time polymerase chain reactions (PCRs) using Assays-on-Demand (Hs00747379_m1 for ID2; Hs00413032_m1 for E2A; Hs00277134_m1 for PAX5; Hs00174333_m1 for CD19; Hs00233566_m1 for CD79A; and Hs00179459_m1 for BLNK; Applied Biosystems, Darmstadt, Germany) and the ABI7900HT Sequence Detection device.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Derivative Assay, Virus, Transformation Assay

Journal: The American Journal of Pathology

Article Title: Aberrant Expression of ID2, a Suppressor of B-Cell-Specific Gene Expression, in Hodgkin's Lymphoma

doi: 10.2353/ajpath.2006.060020

Figure Lengend Snippet: Double IF for ID2 with E2A, PAX5, or CD79A. ID2 staining is always shown in green, E2A, PAX5, and CD79A are always shown in red. A–C: In all three double IFs, strong nucleolar ID2 staining of HRS cells is shown. E2A, PAX5, and CD79A are detectable in several small bystanders but were below the detection level of our double IF in the HRS cells, also when only the red filter was used for examination (it should be noted that the large numbers of B cells seen in B are not typical for most cases of classical HL). D–F: Coexpression of ID2 with E2A, PAX5, and CD79A in L&H cells is shown. CD79A expression is weaker than in small bystanders. G–I: Coexpression of ID2 with E2A, PAX5, and CD79A in MLBCLs is shown. The expression levels of ID2 and E2A vary significantly between individual cells. Although in some cells, either ID2 (green) or E2A (red) expression was predominant, in other cells, similar staining intensities with both fluorescence dyes were observed (yellow). J–L: Strong E2A, PAX5, and CD79A expression relative to ID2 in DLBCL is shown.

Article Snippet: One-microliter aliquots of the cDNAs were used as templates in real-time polymerase chain reactions (PCRs) using Assays-on-Demand (Hs00747379_m1 for ID2; Hs00413032_m1 for E2A; Hs00277134_m1 for PAX5; Hs00174333_m1 for CD19; Hs00233566_m1 for CD79A; and Hs00179459_m1 for BLNK; Applied Biosystems, Darmstadt, Germany) and the ABI7900HT Sequence Detection device.

Techniques: Staining, Expressing, Fluorescence

Journal: The American Journal of Pathology

Article Title: Aberrant Expression of ID2, a Suppressor of B-Cell-Specific Gene Expression, in Hodgkin's Lymphoma

doi: 10.2353/ajpath.2006.060020

Figure Lengend Snippet: Quantitative analysis of ID2, E2A, PAX5, and CD79A expression in primary lymphomas. For quantitative evaluation of immunofluorescence double staining (ID2/E2A, ID2/PAX5, and ID2/CD79A), staining for each antibody combination was performed in one experiment for all cases analyzed, and expression levels were scored by visual inspection of two investigators from 0 to 3 (no detectable to very strong expression). Average values were calculated from 13 cHL, 9 lpHL, 8 MLBCL, and 10 DLBCL analyzed. A: The average expression values for ID2, E2A, PAX5, and CD79A are shown. B and C: The expression levels of ID2 relative to E2A and PAX5 levels, respectively, are shown. They were calculated using the average expression scores shown in A, which ranged from 0.1 for PAX5 in cHL to 2.6 for ID2 in cHL. D: The CD79A expression values are plotted against the ID2-to-E2A ratios for the various lymphoma types analyzed.

Article Snippet: One-microliter aliquots of the cDNAs were used as templates in real-time polymerase chain reactions (PCRs) using Assays-on-Demand (Hs00747379_m1 for ID2; Hs00413032_m1 for E2A; Hs00277134_m1 for PAX5; Hs00174333_m1 for CD19; Hs00233566_m1 for CD79A; and Hs00179459_m1 for BLNK; Applied Biosystems, Darmstadt, Germany) and the ABI7900HT Sequence Detection device.

Techniques: Expressing, Immunofluorescence, Double Staining, Staining

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 5 | Wdr1 is important for cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells that had been transfected with control (Ctrl) siRNA or Wdr1 siRNA were added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC, defined as >90% of the total Ag fluorescence intensity being contained in 1-2 clusters, is graphed. Each symbol on the graph represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n >25 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The ratio is graphed. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of four independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Microscopy, MANN-WHITNEY

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 6 | Cofilin is important for cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells that had been transfected with control (Ctrl) siRNA or cofilin siRNA were added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC is graphed. Each symbol represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n >30 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of three independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Microscopy, MANN-WHITNEY

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 7 | Inhibiting LIMK in A20 D1.3 B cells impairs cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells were pre-treated for 1 h with DMSO or 50 µM LIMKi3 before being added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC is graphed. Each symbol represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n > 67 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of three independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Expressing, Microscopy, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) Schematic representation of a 3-plex SILAC approach for profiling phosphorylation dynamics in resting and BCR-stimulated DG75 cells. DG75 cells were cultured in SILAC medium as indicated and were left untreated, or were BCR-stimulated, for 2, 5, 10, or 20 min. Daudi cells were stimulated for 2 and 10 min. Lysates were mixed in a 1:1:1 ratio and digested with trypsin. Resulting phosphopeptides were enriched by either SCX/TiO2 chromatography (global phosphoproteome analysis; GPome) or phosphotyrosine immunoprecipitation (pY-IP; pYome analysis), and analyzed by LC-MS/MS. For analysis of protein expression levels, proteins were separated by 1D-PAGE, digested with trypsin, and analyzed by LC-MS/MS (see SI Materials and Methods for details). (B) Schematic representation of a 2-plex SILAC approach for profiling phosphorylation changes upon inducible CD79a knockdown or upon SYK inhibition. DG75 cells were cultured in SILAC medium and treated as indicated. Lysates were processed as described in A. (C) DG75 and Daudi cells were loaded with the ratiometric Ca2+-chelator INDO-1-AM and subjected to BCR-induced Ca2+ flux analysis by flow cytometry.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Cell Culture, Chromatography, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: Tonic BCR signaling. (A) CD79a shRNAs are toxic for BL cell lines. The figure shows the fraction of GFP-positive, shRNA-expressing cells relative to the GFP-negative, shRNA-negative fraction at the times indicated (normalized to day 0). Data are representative of three experiments. (B) CD79a and actin immunoblots of lysates derived from DG75 cells that were treated with doxycycline for 18 h to express either unspecific shRNAs (Control) or shRNAs targeting CD79a. (C) BCR cell surface expression in DG75 control cells or CD79a knockdown cells was monitored by flow cytometry 18 h after shRNA induction. (D and E) Unsupervised clustering analysis of all p-sites that were regulated upon BCR stimulation/CD79a knockdown/SYK inhibition. Values for each p-site (row) in all conditions (columns) are colored based on the z-score of the log2-transformed SILAC ratios.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: shRNA, Expressing, Western Blot, Derivative Assay, Flow Cytometry, Inhibition, Transformation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) BCR cell surface expression was monitored by flow cytometry (red line, DG75; blue line, Daudi). (B) Scatter plots showing the fold-change of p-sites on mainly serines/threonines (GPome, Left) and mainly tyrosines (pYome, Right) as determined by quantitative MS upon BCR stimulation versus CD79a knockdown and SYK inhibition. Selected phosphorylated proteins and p-sites are highlighted.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Expressing, Flow Cytometry, Inhibition

Journal: Genes, chromosomes & cancer

Article Title: Reduced Dermatopontin Expression Is a Molecular Link Between Uterine Leiomyomas and Keloids

doi: 10.1002/gcc.20035

Figure Lengend Snippet: Microarray Analysis of Leiomyoma versus Myometrium

Article Snippet: Overall, the expression pattern suggested that leiomyomas were composed of cells of a myofibroblast phenotype. table ft1 table-wrap mode="anchored" t5 caption a7 Gene Fold expression Leiomyoma : Myometrium Hormone-related genes Estrogen receptor β 0.71 (− 1.41) CREBBP 0.74 (− 1.35) Estrogen receptor α 0.75 (− 1.33) Growth Hormone Receptor 0.83 (− 1.21) Progesterone Receptor 0.98 (− 1.02) Prolactin Receptor 1.02 SRC1 1.47 P300-CBP 1.52 Extracellular-matrix-related genes Caldesmon 0.19 (−5.21) Dermatopontin 0.23 (−4.26) Tight Junction Protein-1 0.36 (−2.78) Decorin, Variant C 0.44 (−2.25) Desmin 0.78 (− 1.29) Decorin, Variant A 0.85 (−1.17) Decorin, Variant B 1.43 P311 3.68 Versican 5.79 Cell signaling-related genes Vascular endothelial growth factor 0.26 (−3.92) Transforming growth factor β1 0.71 (− 1.4) Interleukin-4 Receptor 0.92 (− 1.09) Transforming growth factor β3 3.00 Open in a separate window Data presented as leiomyoma expression / myometrium expression.

Techniques: Microarray, Expressing, Variant Assay

Journal: Biomolecules

Article Title: Effects of GHR Deficiency and Juvenile Hypoglycemia on Immune Cells of a Porcine Model for Laron Syndrome

doi: 10.3390/biom13040597

Figure Lengend Snippet: Lymphocyte populations, lymphocyte subpopulations and proliferative capacities of PBMC of WT and GHR -KO pigs. ( A ) Percentage of lymphocytes in the leukocytes of wild-type (WT) (black dots, n = 7) and growth hormone receptor knockout ( GHR -KO) pigs (blue dots, n = 6). Lymphocyte percentages were not significantly different between WT and GHR -KO pigs. ( B – I ) Lymphocyte subpopulations of WT (black dots, n = 16) and GHR -KO pigs (blue dots, n = 15). Proportions of ( B ) CD79a + B cells, ( C ) CD3 + T cells ( D ) CD8α + CD3 − NK cells, ( E ) CD8α + CD3 + T cells, ( F ) SWC5 + γδ T cells, ( G ) CD4 − CD8α + lymphocytes, ( H ) CD4 + CD8α + activated/memory T cells did not significantly differ between WT and GHR -KO pigs. CD4 + CD8α − cells ( I ) significantly differed between WT and GHR -KO pigs. ( J – M ) Proliferative capacity of peripheral blood mononuclear cells (PBMCs) of WT (black dots, n = 10) and GHR -KO pigs (blue dots, n = 10) after polyclonal stimulation with ( J ) pokeweed mitogen (PWM), ( K ) concanavalin A (ConA), ( L ) phytohaemagglutinin-L (PHA-L) and ( M ) M. paradisiaca lectin (BanLec) in vitro revealed similar proliferative capacity of WT and GHR -KO PBMCs. Data are shown as mean ± SD; ns = not significant, * p ≤ 0.05.

Article Snippet: Staining of 3 × 10 5 cells per well was performed with mouse anti-human CD79a monoclonal antibody (mab) (clone HM57, IgG1; Bio-Rad AbD Serotec, Puchheim, Germany; 1:100; cross-reactive to pig [ ]) for the identification of B cells (WT: n = 10; GHR -KO: n = 10).

Techniques: Knock-Out, In Vitro

Journal: bioRxiv

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation

doi: 10.1101/2022.11.19.517148

Figure Lengend Snippet: Mesenchymal stromal cell (MSC) isolation and characterization. (a) Illustration of the isolation process. (b) MSCs migrating out from the tissue. (c) MSCs during the expansion phase (Passage 1 to 4). (d) Surface marker expression for P4 MSCs. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (e) P4 MSCs could be differentiated into adipocytes and osteocytes

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Marker, Expressing

Journal: iScience

Article Title: Human adult, pediatric and microtia auricular cartilage harbor fibronectin-adhering progenitor cells with regenerative ear reconstruction potential.

doi: 10.1016/j.isci.2022.104979

Figure Lengend Snippet: Figure 2. Expression of putative stem cell markers Using flow cytometry, expression of mesenchymal stromal cell specific markers was measured. Data are represented as mean +/- SEM. Individual data points are also shown. High percentages of cells positive for CD90, CD105, and CD73 were found in adult, pediatric, and microtia populations at passage 4. All populations exhibited a low percentage of expression of a panel of surface markers. This negative marker cocktail consisted of CD11b, CD34, CD45, CD79a, and HLA-DR.

Article Snippet: Antibodies Collagen Type I Abcam ab138492; RRID:AB_2861258 Collagen Type II DSHB II-II6B3; RRID:AB_528165 Goat Anti-Mouse HRP DAKO p0447 Elastin Abcam Ab9519; RRID:AB_2099589 Streptavidin conjugated with HRP DAKO P0397 CD45-PE Mouse IgG1 Clone 2D1 R&D Systems FAB1430P; RRID:AB_2237898 CD34-PE Mouse IgG1 Clone QBEnd10 R&D Systems FAB7227P; RRID:AB_10973177 CD11b-PE Mouse IgG2B Clone 238446 R&D Systems FAB16991P; RRID:AB_416844 CD79A-PE Mouse IgG1 Clone 706931 R&D Systems FAB69201P HLA-DR-PE Mouse IgG1 Clone L203 R&D Systems FAB4869P; RRID:AB_1151931 HRP-conjugated EnVision+ for Rabbit DAKO K4010 CD90-APC R&D Systems FAB7335A CD73-CFS R&D Systems 5795-EN CD105-APC Abcam N/A

Techniques: Expressing, Cytometry, Marker