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: 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: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ reduces cSMAC formation and proximal BCR signaling at the immune synapse. Raji D1.3 B cells were transfected with either control siRNA or GMFγ siRNA and added to COS-7 APCs expressing the mHEL-HaloTag Ag (magenta). The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79, cyan). The B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images from one of five independent experiments. Scale bars: 5 μm. (B) The total fluorescence intensity of the mHEL-HaloTag Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each B cell and the median values were calculated for each time point. Each symbol on the graph represents the median value for the GMFγ knockdown cells, expressed as a percent of the median value for the control siRNA-transfected cells for the same time point in the same experiment. The differently shaped symbols represent five independent experiments. Paired t -tests were used to calculate p -values. (C) The percent of cells that had formed a cSMAC, defined as > 90% of the total Ag fluorescence intensity being contained in one or two clusters, is graphed. The different symbols represent independent experiments. Paired t -tests were used to calculate p -values. (D) The total fluorescence intensity of pCD79 that was present in clusters at the B cell-APC contact site was quantified for each B cell. The left panel shows representative data from one experiment. Each dot is one cell. n > 31 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. The Mann-Whitney U -test was used to calculate p -values. The right panel shows the results from five independent experiments, presented as in (B) , with n > 30 cells per condition in each experiment. Each symbol represents a single experiment in which the median pCD79 fluorescence intensity for GMFγ-depleted cells is expressed as a percent of the corresponding median value for the control cells. Paired t -tests were used to calculate p -values. (E) For each B cell represented in (D) , the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of the clustered mHEL-HaloTag Ag. The median (blue line) and interquartile ranges (black box) are shown. The data are presented as in (B , D) . **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05; ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Staining, Microscopy, Fluorescence, Knockdown, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t -tests were used to calculate p -values. ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot

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