Figure 2. Reporter gene assay to measure the neutralization of mWnt1 activity by wildtype and Sclerostin variants.

HEK293TSA cells stably transfected with the Wnt-responsive luciferase reporter construct SuperTOPFlash were transfected with an expression construct for murine Wnt1. After 48 h the cells were stimulated with serial dilutions of wildtype (WT) Sclerostin or variants thereof. (A) Overview of the efficiency of the Sclerostin proteins to neutralize Wnt1 driven luciferase expression (IC₅₀ values are shown as bar diagram). The right panel shows a magnification of the data shown in the left panel. Data represents means with standard deviations (SD) of at least three independent experiments. *: P<0.05, **: P<0.01, ***: P<0.001 (student's t-test with data obtained for WT Sclerostin). (B–D) Measurements showing the dose-dependency of selected mutant Sclerostin proteins. IC₅₀ values of these experiments are included in the overview shown in (A). Measurements were done in duplicate. (E) Reporter gene assay using supernatants of HEK Freestyle cells expressing Sclerostin mutants C84AC142R, ΔLoop or wildtype Sclerostin. Data points represent duplicates. To highlight the location of the mutation in the Sclerostin structure the same color-coding as in Figure 1 is used.