Supplementary Figure 2 from Chromatin Helicase CHD6 Establishes Proinflammatory Enhancers and Is a Synthetic Lethal Target in FH-Deficient Renal Cell Carcinoma

Supplementary Figure 2. FH deficiency accumulates CHD6 via inactivating Keap1, related to Figure 2&3. A. WB and co-IP analysis indicating no interactions between VHL and CHD6 proteins in WCLs of ACHN cells. B. WB analysis (left) and RT-qPCR (right) indicated the CHD6 protein or mRNA levels in control and VHL-depleted RCC cells. C. Schematic illustration of Keap1 deletion mutants. The binding capacity of Keap1 to CHD6 is indicated with the symbol. D. Western blots showing in vitro ubiquitination assays conducted by incubating the reconstituted Keap1–CUL3–RBX1 E3 ubiquitin ligase complex with E1 and E2 enzymes, ubiquitin and GST-CHD6 at 30 °C for 2 h. E. Aligning ETGE motif sequence in CHD6 and other known Keap1 substrates. F. Western blots of WCLs and co-IP samples of anti-FLAG antibody from ACHN cells transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h. G. Western blots of WCLs from ACHN cells transfected with the indicated plasmids. H. Cells infected with indicated plasmids for 48 h and then treated with 50 μg/mL cycloheximide (CHX) and harvested at different time points. Quantified data were shown. I. Western blot showing the products of in vivo ubiquitination assays from ACHN cells infected with lentivirus expressing FH-specific shRNA or control for 48 h. RT-qPCR analysis (right) showing the Keap1 mRNA levels in indicated samples. J. Western blot showing the products of in vivo ubiquitination assays from UOK-262 cells with or without FH overexpression. RT-qPCR analysis (right) indicating the Keap1 mRNA levels. K. Chemical reaction process showing the generation of D2-DMF. DMAD, dimethyl acetylenedicarboxylate; PPh3, triphenylphosphine; D2O, heavy water (2H2O); THF, tetrahydrofuran. L. FH-WT cells were treated with increased DMF, and fumarate levels were detected. M. WB analysis showing Keap1 expressions in cells treated with or without DMF. N. Decreased fumarate levels were detected in control and FH-overexpressing UOK-262, Caki-2 (D238 H), and Caki-2 (E378K) cells. C1 and C2 indicate the different clones. O. Western blotting assays and Co-IP analysis showing the Keap1-CHD6 interactions in UOK-262 cells. P values were calculated using a two-tailed unpaired t-test (B, I, J, L, N). *p < 0.05, **p < 0.01, and ***p < 0.001. ns, no significance.