Figure 1

MED15 positively regulates HIF transcriptional activity.A and B, expression levels of HIF target genes in clinical cancer samples from the TCGA cohorts in the MED15 low expression group (bottom 25%) and high expression group (top 25%) are shown. ∗∗∗∗p < 0.0001 by Student's t test. C–E, luciferase reporter assays in HCT116 cells exposed to 21% O₂ or 1% O₂ for 24 h (C) or transfected with empty vector (EV), HIF1α (D), or HIF2α (E). The Firefly luciferase activity was normalized to the Renilla luciferase activity. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey's multiple comparisons test. F, RT-qPCR analysis of CA9, LDHA, and REDD1 in WT or MED15 KO (KO #17, KO #19) HCT116 cells exposed to 21% O₂ or 1% O₂ for 24 h. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗p < 0.05, ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey's multiple comparisons test. G, Western blot of HIF1α, HIF2α, and CA9 in HCT116 WT or MED15 KO cells exposed to 21% O₂ or 1% O₂ for 24 h. H, Western blot of HIF2α in 786-O WT or MED15 KO cells. I, Western blot of HIF1α and HIF2α in HCT116 cells transfected with EV or HIS-tagged MED15. Cells were exposed to 21% O₂ or 1% O₂ for 24 h. CA9, carbonic anhydrase 9; COADREAD, Colon adenocarcinoma/Rectum adenocarcinoma Esophageal carcinoma; HIF, hypoxia-inducible factor; KIPAN, Pan-kidney cohort; LDHA, lactate dehydrogenase A; MED15, Mediator complex subunit 15; ns, not significant; REDD1, protein regulated in development and DNA damage response 1.