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. 2022 Mar 30;41(18):2638–2650. doi: 10.1038/s41388-022-02279-w

Fig. 5. NEU1 is differentially overexpressed upon EHMT2/G9a inhibition and alters mechanisms of metastatic process in vitro.

Fig. 5

A Venn diagram analysis of overlapping DEG with respect to control conditions, between CADO-ES and RM82 cell lines. B Immunoblot of the main DEG protein after 24 h of BIX10294 treatment at IC50 or IC90 concentrations in EWS cell lines. Relative quantification is shown with respect to the control. C, D EHMT2 expression was silenced by two siRNA constructs in EWS cell lines. Relative quantification of mRNA expression levels of EHMT2 and NEU1 genes at 48 or 72 h after siRNAs transfection (upper bars graphics). Western blot validation of G9a and NEU1 protein expression according to the silencing level in EWS cell protein extracts (lower panels). E, F ChIP-qPCR analysis of G9a (left graphics) and H3K9me2 mark (right graphics) binding levels at NEU1 promoter region in CADO-ES and RM82 EWS cell lines treated with BIX01294 (IC50 concentration). % Input indicates enrichment ratio of immunoprecipitated samples relative to input. The promoter of transcriptionally active EWSR1-FLI1 target gene AURKB was used as binding-negative control. The IgG antibody was used as a control for unspecific binding in ChIP assays. (G, H) Migratory capacity analysis by wound healing assay at 24 h after EWS cell lines were transfected with the NEU1 ectopic overexpression plasmid. Bar graphs show the percentage of migratory cells is shown for each drug treatment with respect to the control in both cell lines. Representative images are shown for the beginning and end points of the assay (right panels). I, J Invasion ability analysis of Transwell migration assay at 48 h after EWS cell lines transfected with NEU1 ectopic overexpression plasmid. Percentage of migratory cells is shown for each drug treatment respect to the control in both cell lines on bar graphics. End point representative images are shown (right panels). K, L Clonogenic capacity analysis at 15 days after EWS cell lines were transfected with NEU1 ectopic overexpression plasmid. Bar graphs show the percentage of total colonies for each drug treatment with respect to the control in both cell lines. Right, representative images of plate captures at the end of the experiment. *p < 0.05; **p < 0.01; ***p < 0.001.