Fig. 2.

HIF2α expression is regulated by canonical Notch signaling, but not through direct transcriptional activation. a Schematic illustration of the NERT2 system where Notch1 ICD nuclear localization and thus Notch signaling is activated by addition of 4-OH tamoxifen (TMX). b, c HIF2α and NRARP mRNA expression after Notch signaling activation by 50 nM TMX in DAOY cells transiently (b) or stably (c) expressing NERT2, in combination with expression of dnMAML (c). d Western blot analysis of CSL levels in CRISPR/Cas9-inactivated (CSL^-/-) and control (CSL^+/+) DAOY cells (left), and with HIF2α mRNA expression upon Notch1 ICD expression in these cells (right). e Analysis of CSL binding to the promoters of HIF2α (EPAS1; top and lower left) and HES1 (lower right) under control or Jagged1-stimulating conditions (Notch on). No specific CSL binding was recorded in the HIF2α promoter, whereas CSL binding to an established CSL-binding site located approximately 60 bp of the HES1 transcription start site was recorded following Notch activation. f Analysis of a HIF2α promoter-luciferase reporter construct upon cultivation of MDA-MB-231 cells on immobilized Jagged1 ligand (Jag1-Fc) or Fc fragments. g–j Analysis of HIF2α (g, i) and NRARP (h, j) mRNA expression in DAOY cells transiently expressing NERT2 in combination with TMX treatment to activate Notch signaling. Cells were treated with 10 µg/mL cycloheximide (CHX) for 8 h to block translation (g, h) or 1 µg/mL actinomycin D (A.D.) for 8 h to block transcription (i, j). Values are significant at *** p < 0.001, **p < 0.01, and *p < 0.05. Graphs represent an average of at least three independent experiments