FIGURE 5 from Targeting the Clear Cell Sarcoma Oncogenic Driver Fusion Gene <i>EWSR1::ATF1</i> by HDAC Inhibition

<p>SOX10 regulates the expression of EWSR1::ATF1. <b>A,</b> CCS cells were treated with 0, 0.3, 1.0, 3.0, or 10.0 μmol/L vorinostat for 24 hours. SOX10 protein expression levels were estimated via Western blotting. Loading controls are identical to those shown in <a href="#fig4" target="_blank">Fig. 4A</a>. <b>B,</b> CCS cells were treated with 0 or 3 μmol/L vorinostat for 24 hours. <i>SOX10</i> mRNA expression levels in CCS cells were quantified using qPCR (normalized to GAPDH; <i>n</i> = 3). <b>C,</b> EWSR1::ATF1 protein expression levels of MP-CCS-SY and SU-CCS1 cells 48 hours after SOX10 knockdown were detected via Western blotting. <b>D,</b><i>EWSR1::ATF1</i> mRNA expression levels of MP-CCS-SY and SU-CCS1 cells 48 hours after SOX10 knockdown were quantified using qRT-PCR (normalized to GAPDH; <i>n</i> = 3). <b>E,</b> Genome browser screenshot of the epigenome maps at EWSR1. Tracks visualize CUT&RUN-seq data for SOX10 (<i>n</i> = 2), and scATAC-seq data are shown for treatments with 3 μmol/L vorinostat or vehicle for 24 hours. Blue highlights indicate the inserted DNA regions used in the reporter assay. <b>F,</b> Relative EWSR1 promoter activity of MP-CCS-SY and SU-CCS1 cells 48 hours after SOX10 knockdown was measured with a reporter assay (<i>n</i> = 3). <b>G,</b> Proliferation of CCS cells after SOX10 knockdown was measured using a WST-8 assay during 1–4 days of culture. <b>H,</b> EWSR1::ATF1 mRNA expression levels of MP-CCS-SY and SU-CCS1 cells 48 hours after SOX10 overexpression. <b>I,</b> Relative EWSR1 promoter activity of MP-CCS-SY and SU-CCS1 cells 24 hours after SOX10 overexpression was measured with a reporter assay (<i>n</i> = 3). <b>J,</b> Proliferation of CCS cells after SOX10 overexpression was measured using a WST-8 assay during 1–4 days of culture. <b>K,</b> MP-CCS-SY and SU-CCS1 cells after SOX10 knockdown or not were treated with vorinostat at 1 μmol/L. Relative cell viability was assessed after 48 hours by WST-8 assay. <b>L,</b> MP-CCS-SY and SU-CCS1 cells after SOX10 overexpression or not were treated with vorinostat at 1 μmol/L. Relative cell viability was assessed after 48 hours by WST-8 assay. <b>M,</b> MP-CCS-SY and SU-CCS1 cells after SOX10 overexpression or not were treated with vorinostat at 1 μmol/L. Relative <i>EWSR1::ATF1</i> mRNA expression levels were quantified using qRT-PCR (normalized to GAPDH; <i>n</i> = 3). Data in B, D, F–M are means ± SDs. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ns, nonsignificant (Student <i>t</i> test).</p>