Fig. 6: So(d18:1) suppress the binding of HIF-2α and ARNT.

A HRE-based luciferase assay in HEK293T transfected with HIF-2α, followed by treatment of PT2385, So(d18:1), So(d16:1), So(d20:1), Sa(d18:1) (n = 3 biologically independent samples). B HRE-based luciferase assay in HEK293T transfected with HIF-1α, followed by treatment of PX-478, So(d18:1) (n = 6 biologically independent samples). C Schematic and experimental representation of PT2385 or So(d18:1) mediated HIF-2α-ARNT interaction by pG5GAL4 luciferase assay followed by pBIND-HIF-2α and pACT-ARNT transfection of HEK293T cells (n = 5). D Co-immunoprecipitation for ARNT and HIF-2α in HEK293T cells transfected with ARNT and the oxygen-stable HIF-2α triple mutant (HIF-2α™) plasmid treated with control solvent, So(d18:1) or PT2385, PT2385 and So(d18:1) could inhibit HIF-2α to bind to ARNT. This experiment was repeated 3 times independently with similar results. E Co-immunoprecipitation for ARNT and HIF-1α in HEK293T cells transfected with ARNT and the oxygen-stable HIF-2α triple mutant (HIF-2α™) plasmid treated with So(d18:1) (20 μM) or TAY-cyclo (20 μM). This experiment was repeated 3 times independently with similar results. F Molecule docking prediction of So(d18:1) binding sites in HIF-2α PAS-B domain. G schematic diagram of site missense mutation experiment. PT2385 and So(d18:1) could inhibit normal HIF-2α transcription ability but not HIF-2α with missense mutations. (n = 6 biologically independent samples). H Cpt1a promoter rHRE constructs plasmid were co-transfected with HIF-2α™ followed by control solvent or So(d18:1) treatment. So(d18:1) could inhibit HIF-2α binding ability to rHRE. (n = 3 biologically independent samples). Data are the means ± s.e.m. A–C, G (WT), statistical analysis was performed using One-way ANOVA. G(mutant), statistical analysis was performed using Kruskal–Wallis test with Dunn’s test. H Statistical analysis was performed using two-tailed Student’s t-tests.