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. 2021 Sep 16;12:5356. doi: 10.1038/s41467-021-25553-z

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© The Author(s) 2021

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. 2 — a Time-course knockdown of EWSR1-FLI1 in A673/TR/shEF1 EwS cells harboring a Dox-inducible shRNA against EWSR1-FLI1 and analysis of PRC1 and EWSR1-FLI1 expression by qRT-PCR in vitro. Dots represent means and whiskers SEM, n = 4 biologically independent experiments. b Analysis of PRC1 expression by IHC in xenografts derived from A673/TR/shEF1 and A673/TR/shCtrl cells with/without Dox-treatment for 96 h in vivo. Data are displayed as individual dots. Horizontal bars represent the median, n indicates the number of biologically independent experiments/cell line: A673/TR/shCtrl n = 9, A673/TR/shEF1 n = 5; scale bar = 200 µm. c Integrative genomics view (hg19) of the PRC1 locus from data of A673 and SK-N-MC cells being transfected with shRNAs targeting either GFP (shGFP; negative control) or EWSR1-FLI1 (shEF1). d Luciferase reporter assays in A673/TR/shEF1 cells with/without knockdown of EWSR1-FLI1 (Dox+/−) 72 h after transfection. Data are displayed as individual dots. Horizontal bars represent means and whiskers SEM, n = 6 biologically independent experiments. e Analysis of PRC1 expression by qRT-PCR (left) and viable cells (right) in TRE-regulated dCas9-KRAB A673 and RDES cells transduced with sgRNAs against the PRC1-associated GGAA-mSat and GFP sgRNAs (negative control) 15d after addition of Dox (2 µg/ml). Data are displayed as individual dots. Horizontal bars represent means and whiskers SEM, n = 6 biologically independent experiments. f Analysis of PRC1 expression by qRT-PCR (left) and viable cells (right) in wildtype (wt), CRISPR Cas9-edited negative control (NC), and CRISPR Cas9-initiated HDR edited (insertion of 24-GGAA-repeats) A673 cells 72 h after seeding. Data are displayed as individual dots. Horizontal bars represent means and whiskers SEM, n = 8 biologically independent experiments. g Relative crosslinking frequencies observed in CRISPR Cas9-initiated HDR edited PRC1-associated GGAA-mSat knockout (KO) A673 EwS cells are shown in gray and insertion of 24-GGAA-repeats in dark red. Gray shading indicates the position and size of the analyzed fragments, and red shading the ‘vantage point’ fragment harboring the PRC1-associated GGAA-mSat. The highest crosslinking frequency value in the graph was normalized and set to 1 in each replicate. Data are mean and SEM, n = 4 biologically independent experiments. Two-sided Mann-Whitney test in all panels.