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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. 9 — a Experimental setup: BM-derived Nsd1^−/− erythroblasts were transduced with either pLMP-empty-shRNA-GFP-Puro (Ctrl-shRNA) or pLMP-Ski-shRNA-GFP-Puro (Ski-shRNA) in maintenance medium, sorted for GFP, and selected with Puromycin for 2 days before induced differentiation and analysis. b Representative images of Wright Giemsa-stained cytospin preparations of control (Ctrl-shRNA, left panel) and Ski shRNA (Ski-shRNA), right panel) transduced Nsd1^−/− BM-derived erythroblasts after 2 days in differentiation medium. The small insets show the cell pellets before analysis. These data illustrate one of three experiments (×1000, size bar = 10 μM). c Growth of Nsd1^−/− BM-derived erythroblasts transduced with Ski- (Ski-shRNA, blue line) or control shRNA (Ctrl-shRNA, black line) grown for 4 days in differentiation medium. Nucleated living cells were counted by Trypan blue exclusion (n = 3 per group). P value > 0.05 for all time points. d Fraction of Ter119⁺ cells (%) of Nsd1^−/− BM-derived erythroblasts transduced with Ski- (Ski-shRNA, blue bars) or control (Ctrl-shRNA, black bars) virus grown for 2 days in differentiation medium (n = 3 per group). e Western blot showing SKI and GATA1 protein expression in Nsd1^−/− BM-derived erythroblasts transduced with Ski (Ski-shRNA) or control (Ctrl-shRNA) virus during expansion in maintenance medium (day 0) and following 1–2 days in differentiation medium. Actin was used as a loading control. These data represent one of three experiments. f Total number of colonies counted at day 11 after plating of 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) in MC (M3434) (n = 3 per group). g Total number of cells obtained from 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) after 11 days in MC (M3434) (n = 3 per group). h Percentage of Kit⁺ living cells obtained from 1 × 10⁴ Nsd1^−/− BM-derived erythroblasts expressing either Ski shRNA (Ski-shRNA, blue bar) or control (Ctrl-shRNA, black bar) after 11 days in MC (M3434) (n = 3 per group). P value > 0.05 for all time points. 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 c, d, f, g, h tested with a paired two-tailed t-test.