Fig. 2. HIF-1α binds to LVBU promoter to promote gene transcription of LVBU.

a Upregulation of LVBU in RKO and HCT116 cells under hypoxia condition (1% O₂) was detected by qRT-PCR. HIF-1α expression was detected by western blot at the indicated time points after hypoxia treatment (N = 3 independent samples, **p < 0.01; ***p < 0.001). b The expression of LVBU was detected by qRT-PCR under normoxia or hypoxia condition when HIF-1α was knocked down (N = 3 independent samples, **p < 0.01). c Predicted binding regions (BRs) of HIF-1 α/1β at the promoter (−2000 bp–TSS) of LVBU were shown. The prediction was performed with the website JASPAR. d Luciferase activities were detected by dual luciferase reporter assay after HEK-293T cells transfected with the indicated reporter plasmids under hypoxia condition (1% O₂). N = 3 independent samples, ***p < 0.001. e Predicted binding motifs of HIF-1α at the promoter region (−300 bp- +1 bp) of LVBU were shown. f Schematic drawing of constructs of LVBU promoter inserted into pGL3-basic luciferase reporter plasmid. Luciferase activity was detected by dual luciferase reporter assay after HEK-293T cells transfected with the indicated reporter plasmids (N = 3 independent samples, *p < 0.05; **p < 0.01). g Chromatin immunoprecipitation (ChIP) assays confirmed the binding site of HIF-1α on LVBU promoter in RKO and HCT116 cells (N = 3 independent samples, ***p < 0.001).