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. 2017 Jul 3;36(43):5995–6005. doi: 10.1038/onc.2017.202

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Copyright © 2017 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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MRTFB knockdown antagonizes the transcriptional effects of EWS-FLI1 depletion. (a) Immunoblot of MRTFA and MRTFB knockdown by transient short hairpin RNA (shRNA) transfection with and without concomitant dox-induced (48 h) EWS-FLI1 silencing. Alpha-TUBULIN was used as a loading control. (b) Scatter plot showing effects of serum on MRTFA/B-dependent gene regulation. x axis: sh-MRTFA/B versus sh-Ctrl under starved conditions. y axis: sh-MRTFA/B versus sh-Ctrl under serum-induced conditions. Red dots indicate serum-responsive MRTFA/B-regulated genes at |logFC|⩾0.8 with some examples given. (c) Comparison of effects of MRTFA/B knockdown versus EWS-FLI1 knockdown in A673/TR/shEF and SK-N-MC. Scatter plots showing log2 fold gene expression changes upon EWS-FLI1 knockdown (x axis; dox versus no dox), and upon MRTFA/B knockdown (y axis; sh-MRTFA/B versus sh-Ctrl) alone (EWS-FLI1-high) or in combination with EWS-FLI1 knockdown (EWS-FLI1-low) in A673/TR/shEF (upper panel) and SK-N-MC cell line (lower panel). (d) Inverse gene regulation by MRTFA/B and EWS-FLI1. Venn diagrams of EWS-FLI1-correlated and -anticorrelated genes (green circles) and MRTFA/B-regulated genes under EWS-FLI1-low conditions (yellow circles) in A673/TR/shEF cells. Overlapping area indicates the number of genes that are partially rescued from EWS-FLI1 knockdown effects by MRTFA/B depletion. Arrows indicate direction of gene expression change for EWS-FLI1 knockdown (dox versus no dox; red, |logFC|⩾1, P<0.05), and for combined EWS-FLI1/MRTFA/B knockdown (sh-MRTFA/B versus sh-Ctrl under dox; blue, |logFC|⩾0.7, P<0.05). (e) Heatmap and unsupervised hierarchical clustering of gene expression changes induced by MRTFA/B knockdown under EWS-FLI1-high and -low conditions (cutoff: |logFC|⩾1.5; P<0.05) from two independent experiments. (f) Gene expression effects of specific short interfering RNA (siRNA)-mediated depletion of either MRTFA or MRTFB. Left: representative immunoblot showing the individual siRNA-mediated knockdown of MRTFA and MRTFB on protein level. Right: scatter plots of gene expression (log2 fold) changes induced by EWS-FLI1 (x axis) versus combined MRTFA/B+EWS-FLI1 (y axis) silencing. The inverse correlation is higher for MRTFB (R=−0.66) than for MRTFA (R=−0.36).