Extended Data Fig. 8: Orthogonality of inducible GR2- and ERβ-based INSPIRE components and construction of logic gates by orthogonal TA-INSPIRE.
From: Chemically inducible split protein regulators for mammalian cells
a, Orthogonality of GR2- and ERβ-based INSPIRE components (nSplit and cSplit fragments) was tested in HEK293T cells by co-transfected individual split fragments fused to split luciferase. Orthogonality was assessed by measuring luciferase activity after cell stimulation with indicated synthetic ligands (MOF and 4-OHT) or natural ligands (COR and EST). b, Schematic presentation of the transcriptional activation system used in (c) to simultaneously modulate expression of two reporter genes (BFP and mCit) using orthogonal GR2- and ERβ-based TA-INSPIRE. pMin represents a minimal promoter, and mCit and BFP represent the mCitrine and TagBFP fluorescent protein genes, respectively. c, Conditional activation of reporter genes mCit and BFP after stimulation of the cells with either synthetic ligands MOF (1 µM) and 4-OHT (10 µM) (left) or natural ligands COR (100 µM) and EST (10 µM) (right), detected with fluorescent confocal microscopy. Scale bars represent 100 µm. Microscopic images are representative of two independent experiments (n = 1 biologically independent sample) and four separate observations within the same experiment. d, Construction of functional OR gate, AND gate, and switching between transcriptional activation and repression (ACT/REP) utilizing GR2- and ERβ-based TA-INSPIRE in combination with TALE DNA-binding domain (see schematics in Fig. 4b–d). Cells were stimulated with natural ligands COR (100 µM) and EST (10 µM) as indicated. Results are shown as fold activation in reporter gene expression compared to non-stimulated mock cells transfected with only reporter plasmid. Detailed descriptions of the genetic components and transfection mixtures are provided in Supplementary Table 4. Plots shows the means ± s.d. of n = 8 biological replicates combined from two independent experiments. See Supplementary Table 8 for sample sizes (n) and P values. Source data are provided as a Source Data file.
