Fig. 6

NFS1 is transcriptionally regulated by MYC. a Hallmark enriched pathway analysis showing pathways associated with the expression of NFS1. b, c Gene set enrichment analysis suggesting that NFS1 is positively correlated with MYC pathways. d Q-PCR analysis showing that NFS1 expression is positively correlated with MYC expression in CRC tumor tissues from SYSUCC (n = 115, Pearson’s correlation analysis). e, f Q-PCR analysis (e) and western blotting analysis (f) of NFS1 and MYC expression in HCT116 and 293T cells with control or silenced expression of MYC. g, h Dual-luciferase promoter activity analysis showing the transcriptional activity of NFS1 in HCT116 and 293T cells with MYC downregulation (g) or upregulation (h). i Schematic illustration of the NFS1 promoter containing two main MYC-binding sites (−531 to −495 and −485 to −454). The strategy for mutating the promoter is also shown. j, k Agarose gel electrophoresis (j) and Q-PCR (k) assay after ChIP analysis showing the occupancy of MYC on the NFS1 promoter (region −478 to −338) in HCT116 and 293T cells. l Luciferase promoter activity analysis of NFS1 transcriptional activity in 293T cells overexpressing NFS1 WT or truncation mutant (i). Vinculin was included as a loading control. The data in (e, g, h, k, l) are representative of three independent experiments and presented as the mean ± SD. The P values in (e) were calculated by two-way ANOVA with Dunnett’s multiple comparisons test, those in (g, h, k, l) were calculated by two-tailed unpaired Student’s t test, and this in (d) was calculated by Pearson’s correlation analysis and chi-square test. **P < 0.01, ***P < 0.001