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. Author manuscript; available in PMC: 2017 Jul 6.
Published in final edited form as: Nature. 2016 Sep 5;539(7627):107–111. doi: 10.1038/nature19795

Figure 1.

Figure 1

PT2399 downregulates HIF target genes. a, Binding of PT2399 (inset) to human HIF2α PAS-B domain assayed by isothermal calorimetry (n=2) b, Immunoblots of anti-ARNT1 immunoprecipitates (IP) and whole cell extracts (input) from ccRCC cell lines treated with PT2399 or DMSO. C = control IP without ARNT1 antibody. c, Heat map of changes in mRNAs in 786-O cells [parental and HIF2α−/− generated with sgHIF2α (guide #6)] treated with 2 μM PT2399 or DMSO for indicated duration (hours). n=1. Examples of HIF target genes are indicated in green. d, Venn diagram of gene sets regulated by PT2399. 249 gene sets were regulated in parental 786-O cells, but not in 786-O HIF2α −/− cells. e, f, Levels of the indicated mRNAs, normalized to beta actin, and then to DMSO controls, in 786-O cells treated with PT2399 at the indicated concentration for 24 hrs (e) or duration with 2 μM PT2399 (f). 786-O cells expressing a HIF2α shRNA or control shRNA were included in (e) for comparison; n=3. g, Immunoblots of 786-O cells treated with PT2399. h, VEGF concentration, normalized to total cellular protein, in media conditioned by 786-O cells treated with PT2399; n=3 biological replicates. Data as mean ± s.e.m. (e and f) or mean ± s.d. (h)