Figure 2. Effects of EWSR1-ETS knockdown on the transcriptome, proteome, and methylome of EwS cell lines.
(A) Representative western blot and dot plot for qRT-PCR (biological replicates, n = 4) for EWSR1-ETS without and with shRNA induction by dox addition. A bar indicates mean; GAPDH was used as a housekeeping protein.19
(B) Dot plot indicating differential expression of non-fused EWSR1, ERG, and FLI1 under EWSR1-ETS-low versus -high (EE-low/high) conditions (n = 4). Only genes with expression of at least 1‰ of the fusion in the EE-high state were considered as expressed and depicted.
(C) Circos plot visualizing genome-wide EWSR1-ETS-binding and effects of EWSR1-ETS knockdown on the transcriptome, proteome, and DNA methylation. Fold changes (FCs) of protein and RNA levels are mean values for all cell lines. Differentially methylated regions (DMRs), ChIP peak sites, and CNVs are displayed stacked for each cell line. Avg., average; EETS, EWSR1-ETS; SE, super-enhancer.
(D) Representative gene set enrichment analysis (GSEA) for EETS co- and anti-regulated genes. NES, normalized enrichment score. See also Tables S3A and S3B.
(E) Weighted correlation network analysis (WGCNA) on the GSEA of EWSR1-ETS-correlated and -anticorrelated genes using average rank of expression FC across each cell line. The depicted network analyses were based on significant gene sets with an NES of a minimum of 2.5 (Table S3B).
(F) Dot plot indicating FCs of the depicted EWSR1-ETS-target mRNA level upon EWSR1-ETS knockdown; lines medians.
(G) Representative micrographs of immunohistochemically stained FLI1, p.MYBL2, PAX7 and SOX6 EwS cell line xenografts. Scale bars, 50 μm.
(H) Scatterplot indicating average expression FC per gene across cell lines at the mRNA and protein level; a red line indicates linear regression (Table S4A).
(I) Scatterplot indicating genes with absolute FC greater than 0.5 at the RNA level in EE-low versus -high condition in more than 6 cell lines and the average delta of the β values of CpG sites, which were uniquely annotated for the promoters of the respective gene.
(J) Before-and-after plot indicating a consistent decrease of the calibrated score for the methylation class EwS (mean value for three replicates each) in the six ESCLA cell lines with highest scores; paired t test.