Fig 1.
Evaluation of Wnt, Hh and Notch luciferase single reporters via luminescence readout.
(A) The Wnt promoter TOP was combined with NLuc (substrate: furimazine). (D) The Hh promoter GLI-RET was backbone switched to obtain GLI-FLuc (substrate: luciferin). (G) For the Notch (Not) reporter construct, the CBF promoter was combined with GLuc (substrate: coelenterazine). All transfections were carried out in 293T cells. B, E, H: Luminescence measurement of cells transfected with the indicated plasmid and co-transfected with either pUC19 control or the indicated inducer plasmid. C, F, I: fold increase in signal for the indicated plasmid upon co-transfection with pUC19 control versus inducer plasmid (C: pWnt3a; F: phGli1; I: phICN1). In B, E and H results are show from a representative experiment; C, F and I are the average of 2–5 independent experiments. (**P≤0.01, ****P≤0.0001, ns = not significant, t-test, n = 4). Normalised RLUs were multiplied with 10 (CBF-GLuc), 100 (TOP-NLuc) or 1000 (FOPFlash, TOPFlash, GLI-RET and GLI-FLuc).
Fig 2.
Evaluation of Wnt, Hh and Notch fluorescent single reporters via flow cytometry.
(A) The Wnt promoter TOP was combined with iRFP. (D) The Hh sensitive promoter PTCH1 (wildtype promoter for the hedgehog receptor PTCH1 [30]) was joined with mTurquoise2 (mT2). (G) For the Notch (Not) reporter construct, the CBF promoter was combined with tdTomato (tdT). Transfection were carried out either in 293T cells (B, C, E, F) or HeLa (H, I). B, E, H: Fluorescence measurement of cells transfected with the indicated plasmid and co-transfected with either pUC19 control (white bars) or the indicated inducer plasmid (grey bars). C, F, I: fold increase in signal for the indicated plasmid upon co-transfection with pUC19 control versus inducer plasmid (C: pWnt3a; F: phGli1; I: phICN1). In B, E and H results are shown from a representative experiment; C, F and I are the average of 3–6 independent experiments. (**P≤0.01, ****P≤0.0001, ns = not significant, t-test, n≥4).
Fig 3.
Evaluation of triple pathway reporter constructs.
(A) The three single luciferase reporter gene plasmids for the Wnt, Hh and Notch pathways were joined by a gateway approach to yield 3P-Luc. (F) 3P-Fluor was generated from the three pathway reporter plasmids with indicated fluorophore reporters. Experiments in B, C, D, E, H and I were carried out in 293T cells. In panel G, 293T cell were used for pWnt3a and phGli1 and corresponding pUC19 control transfections, Hela were used in panel G for phICN1 and corresponding pUC19 control and in panel J. (B): Luminescence measurement of cells transfected with the indicated plasmid and co-transfected with either pUC19 control or the indicated inducer plasmid. All luciferase signals (B-E) were normalized on cell viability using the CellTiter Fluor® assay. Normalised RLUs were multiplied with 0.1 (Wnt-pathway experiments), 1 (Hh-pathway experiments) or 0.01 (Notch-pathway experiments). C-E: Quantification of fold increases for Wnt (C), Hh (D) and Notch (E). (G) Evaluation of compensated 3P-Fluor with a compensation matrix applied. Cells were transfected with 3P-Fluor and co-transfected with either pUC19 control or the indicated inducer plasmid. H-J: Quantification of fold increases of fluorescence positive cells for Wnt (H), Hh (I) and Notch (J). In B and G results are shown from a representative experiment; C, D, E, H, I and J are the average of 3–4 independent experiments. (**P≤0.01, ***P≤0.001, ****P≤0.0001, n.s. = not significant, t-test, n≥3).
Fig 4.
Small molecules evaluation with the novel reporters.
293T cells were transfected with the indicated reporter plasmid and treated with the listed compounds (10 μM). (A) TOP-NLuc; (B) GLI-FLuc, (C) CBF-Gluc. White bars: uninduced conditions (unind.), grey bars: induction with conditioned medium (A: CM, contains Wnt3a) or by co-transfection (B: phGli1, C: phICN1). CHIR = CHIR99021, LGK = LGK-974, LY = LY2090314, Niclo = niclosamide, Soni = sonidegib, Vismo = vismodegib. (**P≤0.01, ***P≤0.001, ****P≤0.0001, n.s. = not significant, t-test, n = 4) Normalised RLUs were multiplied with 100 (TOP-NLuc, GLI-FLuc and CBF-GLuc).
Fig 5.
Cross-activation between Wnt, Notch and Hh studies with 3P-Luc.
293T cells were co-transfected with 3P-Luc and control plasmid pUC19 or the indicated inducer plasmid. Luciferase activity was determined 24h or 48h after transfection. (A) NLuc activity measured with substrate (S) furimazine in the lysate of cells (Wnt activity); (B) FLuc activity measured with substrate luciferin in the lysate of cells (Hh activity); (C) GLuc activity measured with substrate coelenterazine in the supernatant of cells (notch activity); *P≤0.05, **P≤0.01, ***P≤0.001, n.s. = not significant, two sided t-test, average from ≥ 2 independent experiments, n ≥ 6).