Fig. 6

FUBP1 promotes LDHB expression by upregulating HIF1α. (a) SH-EP cells were infected with viruses expressing FUBP1 or with siRNA to knock down FUBP1 for 72 h, followed by western blot analysis. β-Actin served as a loading control. (b) SH-EP cells were infected with siRNA to knock down HIF1α for 72 h or placed in a hypoxic incubator for one week, followed by western blot analysis. (c) SH-EP cells were infected with siRNA to knock down FUBP1 for 72 h or followed by western blot analysis of VHL and HIF1α protein levels. (d) SH-EP cells were infected with FUBP1-overexpressing plasmid or empty vector plasmid, Vhl promoter luciferase reporter plasmid and Renilla luciferase plasmid for 48 h, followed by fluorescence detection. Renilla luciferase served as the transfection control. (e) The candidate FUBP1 binding site, CTTGT (underlined), is shown in the − 1798 to − 1794 region of the Vhl promoter sequence. (f) Chip assays were performed to verify FUBP1 binding to the Vhl promoter. Lane 1: PCR product from immunoprecipitated by normal IgG; Lane 2: PCR product derived from immunoprecipitation by anti-FUBP1 antibody Lane 3: PCR product from immunoprecipitation by anti-Histone antibody; Lane 4: PCR product from input DNA. (g) SH-EP cells were infected with FUBP1-overexpressing plasmid or empty vector plasmid, wild-type Vhl promoter reporter (− 1833 bp~ − 1734 bp) and mutant reporter (the single T to A substitution) for 48 h, followed by fluorescence detection. Renilla luciferase served as the transfection control. Error bars represent the standard deviation (SD); one asterisk, p < 0.05; NS means no significant difference