Fig. 2: Inhibition of HDAC6 activity induces EWSR1-FLI1 downexpression and reduced its oncogenic activity. | Oncogene

Fig. 2: Inhibition of HDAC6 activity induces EWSR1-FLI1 downexpression and reduced its oncogenic activity.

From: Selective inhibition of HDAC6 regulates expression of the oncogenic driver EWSR1-FLI1 through the EWSR1 promoter in Ewing sarcoma

Fig. 2

A Time course experiments (short-, medium-, and long-term) of HDAC6 protein expression was evaluated by immunoblotting using extracts from SKNMC and WE68 EWS cell lines treated with IC50 or IC90 concentrations of BML-281. B Subcellular fractionation and immunoblotting evaluation of HDAC6 localization in both cytoplasm and nucleus fractions. C Dose-dependent evaluation of H4K12 acetylation level in SKNMC and WE68 cell lines exposed to increasing concentrations of BML-281 (0.1, 1, 5, and 10 μM) for 4 h. D RT-qPCR analysis of EWSR1-FLI1 mRNA level in SKNMC and WE68 EWS cell lines treated with BML-281 (IC50 and IC90) in time-course experiments. E Immunoblot assessment of HDAC6 and EWSR1-FLI1 protein expression levels after HDAC6 depletion by two shRNA constructs. F, G RT-qPCR and immunoblot assessment of mRNA and protein expression levels of EWSR1-FLI1 (F, G, upper panels), and EWSR1-FLI1 regulating target genes (F, G, lower panels) after 24 h of BML-281 treatment at IC50 and IC90 concentrations in the SKNMC or WE68 cell line, respectively. H GSEA C2_MSigDB analysis showed the overlap between genes that were significantly repressed or induced by BML-281 in EWS cell lines at EWSR1-FLI1 target genes signatures. I Enrichment plots with the best enrichment based on the |NES| (normalized enrichment score) are shown. Numbers below blots represent densitometric quantification of bands, normalized to endogenous bands and referred to their respective controls (DMSO band from the same time point). *P < 0.05; **P < 0.01; ***P < 0.001.

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