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. 2018 Feb 20;46(7):3412–3428. doi: 10.1093/nar/gky119

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Cooperation of SKI and RUNX1 in gene repression of AML cells. (A) HeLa cells were transfected with empty vector, Flag-tagged SKI and HA-tagged RUNX1 constructs (alone or in combination, as indicated) and harvested 48 h post transfection. Protein extracts were subjected to immunoprecipitation using α-HA antibodies for RUNX1 or isotype control IgG (IgG). Input (3%) and precipitates were analyzed by immunoblot using α-Flag (SKI) and α-RUNX1 antibodies. Size markers (in kDa) are shown on the right. (B) HEK293T cells were transfected with empty vector or Flag-tagged RUNX1 construct and harvested 48 h post transfection. Protein extracts were subjected to immunoprecipitation using α-SKI antibodies for endogenous SKI protein or isotype control IgG (IgG). Input (5%) and precipitates were analyzed by immunoblot using α-SKI and α-Flag (RUNX1) antibodies. Size markers (in kDa) are shown on the right. (C) GST-Pulldowns were performed using recombinant GST-RUNX1 (full-length or deletions, as depicted in the scheme upper panel; RHD = RUNT homology domain, TAD = Transactivation domain) and SKI expressed by in vitro transcription/translation (IVT). Input (20% IVT) and GST pulldown reactions were analyzed by immunoblot using α-SKI antibody (middle panel). Amounts of bead-bound GST-proteins were visualized by Coomassie staining. Asterisks mark the corresponding protein bands (lower panel). (D) Venn diagram shows the overlap of genes associated with SKI/RUNX1 common binding sites and genes differentially expressed upon SKI deletion. ( E) For reporter gene assays, HEK293T cells were transiently transfected with 100 ng of luciferase reporter containing the Elastase2 gene promoter (pGL3-hELA2) and RSV-β-Gal reporter (the latter for normalization) -/+ expression plasmids for RUNX1 (300 ng) and SKI (increasing amounts: 25, 75 and 150 ng). 48 h post-transfection, cells were assayed for luciferase activity, of which mean values (± SD of triplicates) are shown relative to β-galactosidase activity (upper panel). SKI/RUNX1 overexpression was confirmed by immunoblot. CDK2 staining served as loading control (lower panel). (F) HL60 cells stably expressing doxycycline inducible control shRNAs (shCON.1, shCON.2) or shRNAs targeting RUNX1 (shRUNX1.1, shRUNX1.2) were treated with doxycycline for 7 d. RT-qPCR analyses were subsequently performed for a subset of SKI/RUNX1 common target genes. RT-qPCR for RUNX1 confirms the RUNX1 depletion. Values were normalized to GAPDH and presented relative to shCON.1 (mean ± SD of triplicates). (G, H) GSEA comparing genes regulated by Runx1 deletion in a conditional KO mouse model to genes differentially expressed after SKI deletion in HL60 cells (G). GSEA comparing genes regulated by RHD overexpression in mouse (52) to genes differentially expressed after SKI deletion in HL60 cells (H).