Journal: Journal of Cell Science

Article Title: The homeobox gene DLX4 regulates erythro-megakaryocytic differentiation by stimulating IL-1β and NF-κB signaling

doi: 10.1242/jcs.168187

Figure Lengend Snippet: Association of DLX4 expression with increased megakaryopoiesis and decreased erythropoiesis. (A) Heatmap representation of DLX4 mRNA levels in stem, progenitor and mature human hematopoietic cell populations in the gene expression dataset of (GEO Accession no. GSE24759). The labels of the indicated populations correspond to those used in the study of with the exception of the following: HSC (combination of HSC1 and HSC2), CFU-Meg (CFU-MK), early erythroid (ERY1), late erythroid (ERY5), B cell (naïve B cell), T cell (combination of naïve CD4 + and CD8 + T cells). (B) K562 cells were stimulated for 3 days with PMA (left panel) and with ActA (right panel) to induce megakaryocytic and erythroid differentiation, respectively. Shown are mRNA levels of DLX4, ITGA2B and GYPA , relative to the respective levels in unstimulated (unstim.) cells. (C) CD34 + cord blood cells were cultured for 5 days in medium supplemented with TPO cocktail (cktl) (left panel) and in medium supplemented with EPO cktl (right panel) to induce megakaryocytic and erythroid differentiation, respectively. Shown are mRNA levels of DLX4, ITGA2B and GYPA , relative to the respective levels in non-induced (control) cells. (D) CD34 + cells were cultured in medium supplemented with TPO cktl. After 5 days, cells were evaluated by flow cytometry for cell surface staining of CD42a and for intracellular staining of isotype control (left panel) or DLX4 (right panel). The percentages of cells in each quadrant are indicated. (E) CD34 + cells were transduced with GFP-expressing vector control and DLX4 (+DLX4) lentiviruses. Intracellular staining of DLX4 was evaluated by flow cytometry within the gated population of transduced GFP + cells. Solid gray histograms represent staining with DLX4 antibody with mean fluorescence intensities (MFI) indicated. Dotted histograms represent staining with isotype control. (F) Transduced CD34 + cells were sorted for GFP and then seeded in semi-solid medium. After 2 weeks, colonies that originated from 10 4 GFP-sorted cells were scored. Shown in B, C and F are mean±s.d. values of three independent experiments. ** P <0.01, *** P <0.001.

Article Snippet: Sources of antibodies were as follows: DLX4 (Abcam, Cambridge, MA); phosphorylated p65 (Ser536) (Cell Signaling Technology, Danvers, MA); GYPA, CD41 (AbD Serotec, Kidlington, U.K.); CD42a (Miltenyi Biotec, San Diego, CA); IL-1β, CD61, fluorochrome-conjugated secondary antibodies (BD Biosciences, San Jose, CA).

Techniques: Expressing, Gene Expression, Cell Culture, Control, Flow Cytometry, Staining, Transduction, Plasmid Preparation, Fluorescence

Journal: PLoS ONE

Article Title: The role of platelet and endothelial GARP in thrombosis and hemostasis

doi: 10.1371/journal.pone.0173329

Figure Lengend Snippet: GARP and P-selectin expression on 700 μM PAR4-AP ( A ) or 20 μg/mL CRP ( B ) activated murine platelets. Mean fluorescence intensity (MFI) of anti-GARP-APC and anti-P-selectin CD62P-PE are shown. (C) Genotypic analysis of genomic DNA from control C57BL/6, littermates and platelet specific cKO mice. Loxp is 181 bp (fl/fl); wt allele is 111 bp, Cre 250 bp. ( D ) Phenotypic characterization of littermates and cKO mice using flow cytometry. Platelets were labeled with anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD41+ cells: isotype control (black line), littermates (grey line) and cKO (black).

Article Snippet: Expression was determined using fluorescently-labeled antibodies against murine CD45 (30-F11, eBioscience, Vienna, Austria), CD41 (MwReg30), CD31 (390) and GARP (REA 139) (both from Miltenyi Biotec).

Techniques: Expressing, Fluorescence, Control, Flow Cytometry, Labeling

Journal: PLoS ONE

Article Title: The role of platelet and endothelial GARP in thrombosis and hemostasis

doi: 10.1371/journal.pone.0173329

Figure Lengend Snippet: ( A ) Phenotypic characterization of littermates and cKO mice using flow cytometry. Single cells were labeled with anti-CD45-PeCy7, anti-CD31-PE, anti-CD41-FITC and anti-GARP-APC and a histogram was made based on APC intensity of CD45-CD31+CD41- population. Isotype control (black line), littermates (grey line) and cKO (black). ( B ) Genotypic analysis of genomic DNA from control C57BL/6, littermates and endothelial specific cKO mice. Loxp is 181 bp (fl/fl); wt allele is 111 bp, Cre 450 bp.

Article Snippet: Expression was determined using fluorescently-labeled antibodies against murine CD45 (30-F11, eBioscience, Vienna, Austria), CD41 (MwReg30), CD31 (390) and GARP (REA 139) (both from Miltenyi Biotec).

Techniques: Flow Cytometry, Labeling, Control