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. 2018 Aug 1;32(15-16):1008–1019. doi: 10.1101/gad.315192.118

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© 2018 Boulay et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 4. — Silencing a Ewing sarcoma-specific GGAA repeat enhancer does not affect SOX2 expression levels in NCI-H810 lung cancer cells. (A,B) NKX2-2 and SOX2 are expressed at similar levels in lung carcinoma NCI-H810 cells and Ewing sarcoma cells. RT-qPCR (A) and immunoblotting (B) show similar expression levels. EWS-FLI1 is detected only in A673 and SKNMC Ewing sarcoma cells. GAPDH was used as loading control. (C) ChIP-seq for H3K4me3 and H3K27ac (A673 and NCI-H810) and FLI1 (EWS-FLI1) (A673) shows that an EWS-FLI1-bound GGAA repeat near SOX2 is specifically active in Ewing sarcoma. (D) ChIP-qPCR experiments confirm the absence of H3K27ac and the deposition of H3K9me3 at a Ewing sarcoma-specific enhancer near SOX2 in lentivirally transduced NCI-H810 cells with dCas9-KRAB and a sgRNA targeting either the GFP control sequence or the GGAA repeat site near SOX2. (E) RT-qPCR and immunoblotting show that targeting of a Ewing sarcoma-specific repeat enhancer near SOX2 in NCI-H810 cells does not affect SOX2 expression levels in the same experiments. LAMIN A/C was used as loading control. Cells were collected 8 or 10 d after lentiviral transduction. (*) P-value < 0.05. See also Supplemental Figure S5.