Fig. 5: MCL1 is a CIC::DUX4 target gene in CDS cells.

a Western blot analysis of lysates from indicated CDS and EwS tumoroid models. n = 1 independent experiment. b mRNA levels of MCL1 and BCL2, determined by normalized read count analysis of RNA-seq data. Data points indicate individual samples (n = 5 CDS and n = 6 EwS samples). Red dots indicate primary tumors, and black dots indicate tumoroid models. Data are presented as means ± SD. Unpaired two-sided t-test. c ChIP-seq tracks for CIC::DUX4 and H3K27Ac at the MCL1 locus in CDS1 and CDS2 cells. CIC::DUX4 peaks further studied are highlighted in yellow. ChIP-seq data was previously published³⁶. d Log2 fold change of MCL1 mRNA levels in CDS-ZH003 cells after CRISPR interference with the indicated sgRNAs. Data are presented as means ± SD. One-way ANOVA, Tukey’s multiple comparison test, n = 3 independent experiments. e Effect of the indicated shRNA on viability of CDS-ZH003 cells. Cells transduced with indicated doxycycline-inducible shRNA constructs were either treated with doxycycline or left untreated for five days. Live and dead cells were quantified using high content analysis. Plotted are means ± SD. Two-way ANOVA, Tukey’s multiple comparison test, n = 3 independent experiments. p-values were calculated for the live cell fractions. f Western blot analysis of lysates from CDS-ZH003 cells transduced with the indicated shRNA construct and either treated with doxycycline for four days or left untreated. The blot is representative for n = 3 independent experiments. g Quantification of CIC::DUX4 silencing. Western blot bands of the short forms of CIC::DUX4 and CIC were quantified by densitometry, and the CIC::DUX4-to-CIC ratio was calculated. Plotted are means ± SD. Two-way ANOVA, Tukeyʹs multiple comparison test, n = 3 independent experiments. h Quantification of MCL1 protein downregulation. MCL1 and β-Actin Western blot bands were quantified by densitometry and the ratio was calculated. Plotted are means ± SD. Two-way ANOVA, Tukey’s multiple comparison test, n = 3 independent experiments. i Log2 fold change of mRNA levels of MCL1, BCL2 and ETV4 in CDS-ZH003 cells after silencing of CIC::DUX4 with the indicated shRNA. B2M was used as reference gene. Plotted are means ± SD. Two-way ANOVA, Tukey’s multiple comparison test, n = 3 independent experiments. Source data are provided as a Source Data file.