Fig 1: RUVBL1 controls MYC chromatin localization and transactivation activity in EwS. A) RNAseq and GSEA analyses showing changes in expression of the MYC upregulated target gene set in sgCtrl and sgRUVBL1 transduced (day 5) A673-Cas9 cells (two independent sgRNA sequences per group). (Right) Each dot indicates one gene set from the GSEA Molecular Signature Database (MSigDB; total 238 gene sets from the Hallmark and Oncogenic Signature [C6] collections). NES: Normalized enrichment score. B) Growth competition assay of sgCtrl (gray lines; n = 2 independent sgRNA sequences) and sgMYC (purple lines; n = 5 independent sgRNA sequences) in A673-Cas9 cells. C) Cell cycle monitored by EdU incorporation, and D) cellular apoptosis detected by active caspase 3+/DAPI- in A673-Cas9 cells transduced with sgCtrl and sgMYC (n = 3 for each group). E) Western blot of RUVBL1, MYC, histone H4, and GAPDH in A673-Cas9 cells transduced with sgCtrl and sgRUVBL1 (two independent sgRNA sequences per group). F) Co-IP of RUVBL1 (flag-tagged) with RUVBL2 and MYC in HEK293 cells. G) Meta plots (top) and heatmaps (bottom) showing ChIP-seq signal of MYC at TSSs ± 3 kb regions for all genes in A673-Cas9 cells transduced with sgCtrl and sgRUVBL1. H) Profiles of MYC ChIP-seq and (I) ChIP-qPCR at RUVBL1 locus in A673-Cas9 cells transduced with sgCtrl and sgRUVBL1 (n = 3 for each group). J) RT-qPCR of RUVBL1 mRNA in A673-Cas9 cells transduced with sgCtrl and sgMYC (n = 3 for each group). K) Western blot of MYC, RUVBL1, and GAPDH in A673-Cas9 cells transduced with sgMYC (two independent sgRNA sequences per group). L) Model of a feed-forward network between RUVBL1 and MYC in EwS. Data are represented as mean ± SEM. *P < 0.01 compared to sgCtrl by two-sided Student's t-test.