Fig. 6. The effect on Wnt-activated HEK293T cells following treatment with the indicated HiBiT-tagged nTBP-CTPR2n constructs. (A) TOPFLASH reporter assay for HEK293T cells transfected with TCF-Firefly and Renilla reporter gene vectors and an expression vector encoding the constructs listed. For each sample, Luciferase activities were normalised with the Renilla values and the ratio was expressed as relative luciferase activity to the control well transfected with the empty HiBiT vector set at 100% (not shown in the graph). The monomeric constructs, trimeric constructs and controls are shown in different shades of orange, green and blue, respectively. F indicates the foldon motif, H indicates the N-terminal HiBiT tag. Standard deviation was calculated from triplicate sample measurements. The significance of the difference between samples (ns: non-significant, p > 0.05, *p ≤ 0.05 **p ≤ 0.01, ***p ≤ 0.001) was assessed using One-way ANOVA coupled with Dunnett's Multiple Correction test. 1TBP-CTPR2 and 1TBP-CTPR2-F were compared to CTPR2. Multivalent linear and trimeric constructs were compared to 1TBP-CTPR2 and 1TBP-CTPR2-F, respectively. (B) (left) The figure on the left shows the effect on Wnt signaling of treatment with fusogenic liposome-encapsulated 3TBP-CTPR6. Each treatment was with 20 μL of liposomes. In brackets are the concentrations of the proteins used. For each run, data were normalised by the untreated control well, set at 100%. Bars with diagonal stripes correspond to samples treated with liposomes. No Wnt: cells without Wnt pathway activation and not treated with liposomes. Untreated cell: cells not treated with liposomes. FL: empty liposomes. Error bars were obtained from triplicate sample measurements from two independent experiments. The same statistical analysis was performed as in (A), comparing the samples to FL-CTPR6. (B) (right) For comparison, the figure on the right shows the effect on Wnt signaling of hTNKS small molecule inhibitors used interventionally at the indicated concentration. Data were normalised relative to the untreated control well, which was set at 100% (not shown in the graph). Error bars were determined from two independent sample measurements. The same statistical analysis was performed as in (A), comparing the small molecules to DMSO.