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. 2021 May 1;49(9):5038–5056. doi: 10.1093/nar/gkab305

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© The Author(s) 2021. 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 License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — ADD3 pre-mRNA splicing induced by EWS-FLI1 participates in Ewing sarcoma phenotype. (A) Overlap between EWS-FLI1- or RBFOX2-dependent exons in A673/TR/shEF cells and EMT-associated splicing events in H358 human epithelial cells. All pair-wise comparisons are highly significant (P-value < 10^–50). (B) Heatmap of Z-scores of percent of spliced-in (PSI) values from significantly differentially spliced exons in EWS-FLI1^high (A673/TR/shEF cells without Dox treatment) versus EWS-FLI1^low (A673/TR/shEF cells at day 7 of Dox treatment) and in epithelial versus mesenchymal-like ZEB1-expressing H358 cells. (C) Distribution of top significant GO biological process terms from genes containing EWS-FLI1 and RBFOX2-regulated exons. Number of spliced genes regulated by EWS-FLI1 and RBFOX2 in each GO are indicated in brackets. (D) RT- PCR analysis of ADD3 exon 14 splicing. Samples are RNA from A673/TR/shEF cells expressing (-DOX) or not (+DOX) EWS-FLI1 (left) and RNA from A673/TR/shEF cells expressing EWS-FLI1 and transfected with control siRNA (siCTRL) or a specific RBFOX2 siRNA (siRBFOX2). (E) RNA-immunoprecipitation experiments using anti-RBFOX2 antibodies followed by RT-qPCR to detect ADD3 transcripts. Samples are RNA from A673/TR/shEF cells expressing (–DOX) or not (+DOX) EWS-FLI1. Results shown are means ± s.e.m. (n = 3 independent experiments) relative to the –DOX condition. *P < 0.05 by two-tailed unpaired Student's t-test. (F) Immunofluorescence of actin filaments stained with phalloidin (green channel) and DAPI (blue channel) of A673/TR/shEF cells and cells deleted for ADD3 exon 14 genomic region (A673/TR/shEF Δexon14 ADD3). (G) Measurement of cell area of A673/TR/shEF cells and cells deleted for ADD3 exon 14 genomic region (A673/TR/shEF Δexon14 ADD3). Data are represented as mean ± s.e.m. (n > 100 cells). ****P < 0.0001. (H) Three-dimensional type-I collagen multicellular spheroid invasion assay of A673/TR/shEF cells and cells deleted for ADD3 exon 14 genomic region (A673/TR/shEF Δexon14 ADD3). Red dotted lines represent the initial spheroid area. (I) Immunofluorescence of actin filaments stained with phalloidin (green channel) and DAPI (blue channel) of A673/TR/shEF cells treated with DOX for 7 days (EWS-FLI1^low expressing cells) and transfected with either empty vector or vector expressing the ADD3-L isoform. (J) Kaplan-Meier curve of ADD3 exon 14 PSI values in 43 Ewing tumors showing significant differences between tumors expressing high and low levels of ADD3-L isoform. Tumors were separated by k-means clustering. Number of tumors in each subgroup is indicated in brackets.