Fig. 2: dFLASH provides a sensitive readout of three distinct TF pathways.

a–c Three distinct enhancer elements enabling targeting of three different signalling pathways. a Hypoxic response elements (HRE) provide a read out for HIF-1α activation (2OG; 2-oxogluterate, PHD; prolyl hydroxyl domain protein, FIH; factor inhibiting HIF); b Progesterone response elements (PRE), derived from progesterone receptor target genes, facilitate reporting of progestin signalling (FKBP52, HSP90 heat shock proteins, PR progesterone receptor, CoAct transcriptional coactivators) (c) Gal4 response elements (GRE) enable targeting of synthetic transcription factors to dFLASH such as a GAL4DBD-HIFCAD fusion protein that provides a FIH-dependent reporter response (rtTA; reverse tetracycline-controlled transactivator, gal4DBD; gal4 DNA binding domain, HIFCAD; HIF1-α C-terminal transactivation domain). a–c Created in BioRender. Peet, D. (2025) https://BioRender.com/s67t615d–i Flow cytometry histograms and dot plots showing Tomato expression following 48 h treatments of the indicated dFLASH polyclonal reporter cells (d, g) HEK293T HRE-dFLASH; 1 mM DMOG or 0.1% DMSO (ctrl), (e, h) T47D PRE-dFLASH; 100 nM R5020 or Ethanol (ctrl), (f, i) HEK293T dFLASH-synFIH; 1 μg/mL Doxycycline (Dox) and 1 mM DMOG or Dox and 0.1% DMSO (ctrl). Percentages of Tomato-positive ligand-treated population are displayed. j–l Reporter populations as in (d–i) were temporally imaged for 38 h using HCI directly after treatment with (j) 0.5 mM DMOG or 0.1% DMSO, (n = 4 biological replicates per group) (k) 100 nM R5020, 35 nM E2, 0.5 mM DMOG or 0.1% Ethanol (EtOH) (n = 4 biological replicates per group), (l) 0.1% DMSO, 1 mM DMOG, 100 ng/mL Dox and 0.1% DMSO, or 100 ng/mL Dox and 1 mM DMOG (n = 4 biological replicates per group). Data presented as mean ± sem in (j–l). Source data are provided as a Source Data file.