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. 2020 Jun 12;11:2807. doi: 10.1038/s41467-020-16179-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Relative Gata1 mRNA expression levels (1/dCt) in BM-derived erythroblasts from Nsd1^fl/fl (n = 4, black bars) and Nsd1^−/− mice (n = 4, red bars) in maintenance medium (day 0) and after 2 days in differentiation medium. Ct values were normalized to Gapdh expression. b GATA1 protein levels in Nsd1^fl/fl (left panels) and Nsd1^−/− (right panels) BM-derived erythroblasts expanded in maintenance medium (0 h) and in differentiation medium (2.5, 5, and 24 h). LAMIN-A/C was used as immunoblot loading control for nuclear proteins (one out of two experiments). c Number of colonies formed by 5 × 10³ lineage-marker-depleted BM-derived erythroblasts in MC (M3434) from Nsd1^fl/fl (black bars) and Nsd1^−/− mice (red bars) transduced with pMSCV-puro (Ctrl) or pMSCV-mGata1-puro (Gata1) (n = 6 per group). d Ter119 expression (Ter119⁺, in %) in maintenance medium (0 h) and after 2 and 4 days in differentiation medium of Nsd1^fl/fl (black and gray bars) and Nsd1^−/− (red and pink bars) BM-derived erythroblasts transduced with control virus (Ctrl, black and red bars) or Gata1-expressing virus (Gata1, gray or pink bars) (n = 3 per group). e Representative images (one out of two experiments) of Wright Giemsa-stained cytospin preparations and cell pellets (small insets) of Nsd1^fl/fl and Nsd1^−/− BM-derived erythroblasts transduced with control virus (Ctrl) or Gata1-expressing virus (Gata1) after 5 days in differentiation medium (×600, size bars = 10 μm). f HbbA and g Spi1 mRNA levels in BM-derived Nsd1^−/− BM-derived erythroblasts transduced with control virus (Ctrl, black dots) or Gata1-expressing virus (Gata1, red dots) measured 0, 5, 24, 48, and 72 h in differentiation medium. Values are residual ΔCT relative to Gapdh, after adjustment for effect of individual mouse (Tukey test for difference in expression between transductions at 24 h in linear model with interaction between time a and transduction, adjusting for effect of mouse, two-sided, adjusted for multiple comparisons). Values are presented as individual points, bar graphs represent the mean value of biological replicates, error bars as standard error of the mean. Statistical significances in a, c was tested with unpaired two-tailed t-test. Statistical significance in d was tested with either unpaired (Nsd1^fl/fl.Gata1 vs. Nsd1^−/−.Gata1) or paired (Nsd1^−/−.Ctrl vs. Nsd1^−/−.Gata1) two-tailed t-test. Statistical significance in f, g was tested using Tukey test for difference in expression between transductions at 24 h in linear model with interaction between time a and transduction, adjusting for effect of mouse.