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. 2013 May 23;8(5):e64248. doi: 10.1371/journal.pone.0064248

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© 2013 Pencovich et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Left panel; Southern blot analysis of BamH1 digested E14.5 FL cell DNA from Runx1^F ^/F-Pf4-Cre mice (FL) and mature purified FL derived MK^Runx1−/− (FLMK). Middle panel; RT-qPCR analysis of Runx1 expression in MK^Runx1−/− relative to MK^Runx1F/F cells. Data represent the mean ± SD of two independent experiments performed in triplicates. Right panel; Western blot analysis of FL-MK^Runx1L/L (MK^L ^/L) and FL-MK^Runx1−/− (MK^−/−) nuclear extracts using anti-Runx1 antibody. Emerin was used to monitor the amount of loaded protein. (B) FACS analysis of bone marrow (BM) cells derived from Runx1^L ^/L and Runx1^F ^/F-Pf4-Cre mice. Cells were cultured for 3 days with TPO 50 ng ml⁻¹ stained with anti-CD41-PE and anti Ter119-FITC antibodies and analyzed by FACS. (C) FACS analyses of cultured (7 days plus TPO) FL MK^Runx1L/L and MK^Runx1−/− by forward scatter (FSC) Vs side scatter (SSC). (D) Assessment of colony forming unit megakaryocytes (CFU-meg) of E-14.5 FL cells derived from Runx1^L ^/L or Runx1^F/F-Pf4-Cre embryos. Data represent the mean ± SD of two independent experiments performed in triplicates. Increased colony numbers in Runx1^F ^/F-Pf4-Cre, relative to Runx1^L ^/L mice, was statistically significant [P = 0.002]. (E) Analysis of hematopoietic indices in blood of WT, Runx1^L ^/L and Runx1^F ^/F-Pf4-Cre mice. Analysis was carried out using blood samples (n = 5^b or 8^c) obtained by bleeding the orbital sinus: RBCs, red blood cells; WBCs, white blood cells; NEUT, neutrophils; LYMP, lymphocytes; PLAT, platelets.(F) Gene expression alterations in maturating MK lacking Runx1. Genes are plotted based on their expression level (log2 scale) in MK^Runx1−/− vs MK^Runx1L/L. Genes showing 1.5-fold increase or decrease in expression levels are indicated in green or red, respectively. Examples of up/down-regulated genes known to play role in megakaryocytic differentiation and cell proliferation are indicated. (G) Validated expression levels of several Runx1 responsive genes by RT-qPCR using RNA from either FL-MK^Runx1L/L or FL-MK^Runx1−/−. The data represent means ± SD of two experiments performed in triplicates.