Fig. 6: NR5A2 transcriptionally upregulates the expression of ALDH1B1.
A, B Schematic representation of predicted NR5A2 binding sites in the promoter region of human (A) and mouse (B) ALDH1B1, as assessed by the dual luciferase reporter assay. The promoter region spans from −2000 bp to +100 bp relative to the transcriptional start site in human and from −2079 bp to +100 bp in mice. h(m)AP-NBS: NR5A2 binding site within the promoter region of human (mouse) ALDH1B1. (C-D) Evaluation of NR5A2 transcription activity on truncated versions of human (C) and mouse (D) ALDH1B1 promoter regions using a dual-luciferase reporter system. h(m)AP: promoter region of human (mouse) ALDH1B1. 2/3 h(m)AP: The second or third truncation of the promoter region. N = 3/group, ns, not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001. Data are presented as means ± SD. E Schematic representation of NR5A2 binding site mutations in the promoter region of ALDH1B1. F, G Dual-luciferase reporter system employed to analyze the transcriptional activity of NR5A2 on the mutated human (F) and mouse (G) ALDH1B1 promoter region. h(m)AP-mut1/2/3 indicates mutation at the first, second or third predicted binding site was mutant in human (mice). Human: N = 3/group, mouse: N = 6/group, ns not significant, ***P < 0.001, ****P < 0.0001. Data are presented as means ± SD. H Western blotting analysis of ALDH1B1 expression in the LO2-NR5A21in9397-C4 and AML12-Nr5a26in327 cell lines. I RT-qPCR quantification of ALDH1B1 expression in the LO2-NR5A21in9397, LO2-NR5A22ex85 and AML12-Nr5a26in327 cell lines. LO2: N = 3/group, AML12: N = 4/group, *P < 0.05. Data are presented as means ± SD. J, K RT-qPCR assessment (J) and western blotting analysis (K) of ALDH1B1 expression in the LO2 and AML12 cell lines overexpressing NR5A2 isoform1. LO2: N = 6/group, AML12: N = 3/group, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD.
