Fig. 4: IDH1-R132H mutation significantly enhanced the methylation modification level of Ndufa1 promoter.

A GO enrichment analysis was performed on genes in the differentially upregulated promoter region in RBBS sequencing, and the most significantly enriched biological process items were selected for display. B Clones of the BSP products were analyzed by DNA sequencing. 10 clones for each amplicon were sequenced; each row represents the methylation status of an individual sequenced clone. Each circle represents one CpG site, with ● depicting methylated site and ○ depicting unmethylated sites. C, D RT-PCR quantification and WB of NDUFA1 expression relative to β-actin in PTCs isolated from Idh1^WT/WTKsp^Cre and Idh1^WT/MutKsp^Cre mice. n = 6, ****P < 0.0001. E The combination of MSssI treatment and luciferase activity was used to determine whether CpG methylation directly contributes to transcriptional repression of the Ndufa1 gene. Compared to control cells (fragments not treated with MSssI), luciferase activity was significantly downregulated in 293 T cells containing the Ndufa1 promoter reporter construct treated with MsssI, ****P < 0.0001. F When the IDH-R132H overexpression plasmid or empty vector was co-transfected with pGL4-Ndufa1 and renilla luciferase vector into 293 T cells, luciferase activity of Idh1^{WT/ Mut} group was reduced as compared to the Idh1^{WT /WT}group, **P < 0.01.