Fig. 5: TXNRD1 is regulated by USF2 through the transcriptional suppression.

A Venn diagrams show overlaps of putative transcription factors which might bind to the TXNRD1 promoter from three different database. B Relative luciferase activity was detected to analyze the regulation of ten transcription factor in the 2-kilobase human TXNRD1 promoter in HEK293T cells. C HEK293T cells were transiently transfected with ten transcription factors with Flag tag or empty vector for 24 h. Western blot was carried out to confirm the overexpression of indicated transcription factors and detect the protein level of TXNRD1. D Western blot analysis of TXNRD1 and USF2 following transient transfection of Flag-USF2 or vector control in Bel-7402 and Huh7 cells. E The mRNA levels of TXNRD1 were detected by qRT-PCR in Bel-7402 and Huh7 cells transiently transfected with Flag-USF2 or vector control. F Sequences of the canonical USF2-binding E-box motif, putative USF2-binding E-box sites in TXNRD1 promoter, and introduced point mutations used to inactivate the E-box site. G Fragments of the TXNRD1 promoter are shown. Relative luciferase activities of pGL4.17 containing TXNRD1 promoter truncations in Bel-7402 cells exposed to USF2 or control vector were measured by dual-luciferase assays. H Schematic E-box mutations of the TXNRD1 promoter are shown. Relative luciferase activities of pGL4.17 containing E-box site mutations in Bel-7402 cells exposed to USF2 or control vector were measured by dual-luciferase assays. I ChIP analysis of indicated cells immunoprecipitated by anti-USF2 antibody or IgG followed by qRT-PCR using specific primers targeting the USF2-bingding E-box sites in TXNRD1 promoter. Enrichment represent the percentage of input. Data represent the mean ± SEM. ns, not significant; **P < 0.01, ***P < 0.001. P values were calculated by Student’s t test.