Supplementary Figure 4 from Chromatin Helicase CHD6 Establishes Proinflammatory Enhancers and Is a Synthetic Lethal Target in FH-Deficient Renal Cell Carcinoma

Supplementary Figure 4. CHD6 activates NF-κB signaling to potentiate FH-deficient RCC malignancy, related to Figure 5. A. Volcano plot showing differentially expressed genes in UOK-262 cells upon CHD6 knockdown. B. Unsupervised cluster analysis of differentially expressed genes in control and CHD6 knockdown UOK-262 cells. C. ATAC-seq signals showing the profiles of OCRs across the indicated peaks in UOK262 cells with versus without CHD6 knockdown. D. Heatmap exhibiting the significance of transcription factor motifs enriched in accessible loci derived from control and CHD6-KD UOK262 cells. NF-κB motif is dominantly highlighted. E. Venn diagram showing overlapping hits, defined as CHD6-signature, with CHD6 ChIP-seq peaks and changes in OCRs (ATAC-seq) and differentially expressed genes (RNA-seq). F. Western blotting assays and Co-IP analysis showing the altered CHD6-p65 interactions in UOK-262 cells with or without FH restoration. G-H. MTT (G), colony formation (H-left) assays were performed in control and p65-KD UOK-262 cells. Colony formation assays performed in p65-depleted UOK-262 cells with or without CHD6 overexpression (H-right). I. Quantification of colony formation numbers in indicated groups from (H). J. Effects of JSH-23 treatment (1 mg/kg) on UOK262-derived xenografts, as indicated (n = 6 per group, 2-way ANOVA followed by Tukey’s multiple comparisons test). Treatment started when tumors reached 50–100 mm3. K. ChIP-qPCR analysis of CHD6, Pol II-S5P and S2P in the promoter regions of the indicated genes in WT and CHD6-KO UOK-262 cells with restoration of WT CHD6. P values were calculated using 2-tailed Student’s t-test (H, K). *p < 0.05, **p < 0.01, and ***p < 0.001. ns, no significance.