Journal: PLoS ONE

Article Title: Ectopic Runx1 Expression Rescues Tal-1-Deficiency in the Generation of Primitive and Definitive Hematopoiesis

doi: 10.1371/journal.pone.0070116

Figure Lengend Snippet: (A) Undifferentiated ESCs were seeded on OP9 stromal cells (day 0). From day 5 of the culture, the conditions for the different lineages are the same. From day 5 on, different cytokines were added to the cultures and feeding stromal cell layers were changed according to the lineage. (B) qRT–PCR analyses were performed from 8 days differentiated (αMEM/3% SCF) WT, Runx1 −/− , and Tal-1 −/− ESCs out of 3 independent experiments. Bars represent mean ± SD. (C) Representative growth curves of WT ESCs in comparison to Runx1 −/− and Tal-1 −/− ESCs from day 0–18 of the culture (n = 3). Dots show mean ± SD. Development of (D) mesodermal cells, (E) hematopoietic progenitors, (G) erythrocytes and (H) osteoclasts that are differentiated from undifferentiated cells. FACS blots are representative examples of differentiated ESCs. Osteoclasts were identified by staining with TRAP-solution. (F) Apoptosis was evaluated after staining with Annexin-V at day7 of the culture. Flow cytometry profile represents Annexin-V-Cy5 staining in x axis and 7-AAD in y axis. OP9: stromal cell line, DL1: Delta-like 1, ST2: stromal cell line, αMEM: alpha-minimum essential medium, IMDM: iscove’s modified dulbecco’s medium, SCF: stem cell factor, Dex: dexamethasone, VitD 3 : Vitamin D 3 , Epo: erythropoietin, IL: interleukin, Flt3-L: Fms-like tyrosine kinase 3 ligand; SD: standard deviation.

Article Snippet: Cy5 anti-mouse c-Kit (2B8), PE-Cy7 anti-mouse CD45 (30-F11), PE anti-mouse AA4.1 (AA4.1), PerCpCy5.5 anti-mouse Ter119 (TER-119), PE anti-mouse CD71 (R17217), PB anti-mouse Mac1 (M1/70), Cy5 anti-mouse CD19 (1D3), PE-Cy7 anti-mouse B220 (RA3-6B2), biotin anti-mouse Tie2 (TEK4), PE anti-mouse Flk1 (Avas12A1), PE anti-mouse Gr1 (RB6-8C5), PB anti-mouse CD44 (IM7), PerCpCy5.5 anti-mouse CD25 (PC61), PE-Cy7 anti-mouse CD4 (GK1.5), Cy5 anti-mouse CD8 (53-6.7), Cy5 Annexin V and APC-coupled Streptavidin were obtained from eBioscience, San Diego, CA.

Techniques: Quantitative RT-PCR, Staining, Flow Cytometry, Modification, Standard Deviation

Journal: PLoS ONE

Article Title: Ectopic Runx1 Expression Rescues Tal-1-Deficiency in the Generation of Primitive and Definitive Hematopoiesis

doi: 10.1371/journal.pone.0070116

Figure Lengend Snippet: (A) For the development of AA4.1 + CD19 + B220 low B lymphoid cells from day 10 on IL-7 were added to the culture. (B) For the development of CD4 + and/or CD8 + T cells from day 10 on IL-7 were added to the culture and stromal cell layer were changed to DL1-OP9. FACS blots are representative examples of differentiated ESCs. (C) qRT–PCR analyses were performed from differentiated WT, Runx1 −/− , Tal-1 −/− , and Runx1-expressing Tal-1 −/− , and Runx1 −/− ESCs on day 8 of the culture out of 3 independent experiments. Bars represent mean ± SD. OP9: stromal cell line, DL1: Delta-like 1, SCF: stem cell factor, IL: interleukin, Flt3-L: Fms-like tyrosine kinase 3 ligand; SD: standard deviation, CD: cluster of differentiation.

Article Snippet: Cy5 anti-mouse c-Kit (2B8), PE-Cy7 anti-mouse CD45 (30-F11), PE anti-mouse AA4.1 (AA4.1), PerCpCy5.5 anti-mouse Ter119 (TER-119), PE anti-mouse CD71 (R17217), PB anti-mouse Mac1 (M1/70), Cy5 anti-mouse CD19 (1D3), PE-Cy7 anti-mouse B220 (RA3-6B2), biotin anti-mouse Tie2 (TEK4), PE anti-mouse Flk1 (Avas12A1), PE anti-mouse Gr1 (RB6-8C5), PB anti-mouse CD44 (IM7), PerCpCy5.5 anti-mouse CD25 (PC61), PE-Cy7 anti-mouse CD4 (GK1.5), Cy5 anti-mouse CD8 (53-6.7), Cy5 Annexin V and APC-coupled Streptavidin were obtained from eBioscience, San Diego, CA.

Techniques: Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: ROQUIN/RC3H1 Alterations Are Not Found in Angioimmunoblastic T-Cell Lymphoma

doi: 10.1371/journal.pone.0064536

Figure Lengend Snippet: Levels of ROQUIN transcripts determined by gene expression profiling of 17 AITL tumor tissue samples and 2 AITL cell suspensions enriched in tumor cells (≥50%) samples as previously reported . (probe-set 228996_at): ROQUIN transcripts level is slightly higher in enriched tumor cell sample (P = 0,0067 unpaired t-test) (A). ROQUIN mRNA levels determined by quantitative RT-PCR in reactive tonsils; total extract (n = 1), CD4 + (n = 2), CD8 + (n = 2), or CD19 + (n = 2) lymphocytes. Results were normalized by HPRT and compared to reactive purified TFH cells as calibrator: reactive CD4- CD8- and CD19-positive subsets display heterogeneous levels of ROQUIN mRNA (B). ROQUIN mRNA levels [(228996_at) probeset] in purified reactive (n = 12) and neoplastic T FH cells (n = 8). T FH cells were purified from 12 reactive tonsils and 8 AITL lymph nodes, RNA was extracted and whole genome expression was analysed on HG-U133 plus 2.0 Affymetrix GeneChip arrays. Similar levels of ROQUIN transcript are observed (C).

Article Snippet: Tonsil CD4 + , CD8 + T-cells and B-cells were purified by positive selection with antibodies directed against CD4, CD8, and CD19 respectively (Milteny Biotec, Paris, France).

Techniques: Gene Expression, Quantitative RT-PCR, Purification, Expressing