Figure 3

Figure 3

MED15 is a HIF target gene.A, HCT116 cells were exposed to either 21% O₂ or 1% O₂ for 24 h. RNA sequencing was performed to analyze the transcript levels of Mediator complex subunits. B, HCT116 cells were exposed to 21% O₂ or 1% O₂ for 24 h, and Western blot assay was performed with indicated antibodies. C, correlations analysis of MED15 and HIF1α mRNA levels in cancer samples from TCGA is shown. The sample correlation coefficient (R) and corresponding p values are indicated. D, RT-qPCR analysis of MED15 mRNA levels in HCT116 cells exposed to 21% O₂ or 1% O₂ for 24 h. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗∗∗p < 0.001 by Student's t test. E, HCT116 cells were exposed to 1% O₂ for the indicated times, or transfected with HIF1α expression vector, or treated with CoCl₂. Western blot analysis was then performed to evaluate MED15 protein levels. F, Western blot of MED15 protein in HCT116 cells transfected with empty vector or HIF2α expression vector. G, diagram of the MED15 promoter region (6000 base pairs upstream to 1500 base pairs downstream of the transcriptional start site (+1), NCBI reference sequence NC_000022.11: 20501610-20509109) analyzed for potential HREs. H, luciferase assay in HCT116 cells cotransfected with pSV40-Renilla and p2.1, different pGL2 HREs, or pGL2 -3000 ∼ +1 region and then exposed to 21% O₂ or 1% O₂ for 24 h. The Firefly luciferase activity was normalized to the Renilla luciferase activity. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗∗∗∗p < 0.0001 by Student's t test. I, diagram of pGL2 site8 and pGL2 site8-MUT. J, luciferase assay in HCT116 cells cotransfected with pSV40-Renilla and pGL2 site8 or pGL2 site8-MUT and then exposed to 21% O₂ or 1% O₂ for 24 h. The Firefly luciferase activity was normalized to the Renilla luciferase activity. Data are represented as means ± SD. n = 3 biologically independent extracts. ∗∗∗∗p < 0.0001 by Student's t test. HIF, hypoxia-inducible factor; MED15, Mediator complex subunit 15; MUT, mutant; ns, not significant.