Reporter activity was measured with the SelectScreen Cell-based Pathway Profiling Service (Life Technologies) with GeneBLAzer. hsBCL9CT-24 (TFA, HCl, and C2H3O2 salts), hsBCL9CT-35, ICG-001 (Selleck), and LGK-974 (Selleck) dose-dependent inhibition curves were analyzed in Wnt/β-cat signaling pathways via Reporter CellSensor LEF-TCF-bla HCT116 (Invitrogen). All reported data represent triplicate averages.

CellSensor LEF/TCF-bla HCT116 cells contain a β-lactamase reporter gene under the control of the β-cat/LEF/TCF response element stably integrated into HCT116 cells (Life Technologies). LEF/TCF-bla HCT116 cells were shown to constitutively express β-lactamase, which can be further stimulated with mouse Wnt3a. There are seven copies of the LEF/TCF consensus binding sequences, and this cell line can be used to detect agonists and antagonists of the Wnt/β-cat signaling pathway. In the studies reported here, IC50 determination used a standard 10-point titration curve with threefold dilution, according to the best practices for accurate IC50 determination reported in the literature (40). LEF-TCF-bla HCT116 cells were thawed and prepared as described above for the Activator Screen. Thirty-two microliters of cell suspension was added to each well of a 384-well poly-d-lysine assay plate. Cells in assay media were then incubated for 16 to 24 hours in the plate at 37°C/5% CO2 in a humidified incubator. Four microliters of a 10× serial dilution of ICG-001 (control inhibitor starting concentration of 25,000 nM) or other compounds was added to appropriate wells of the plate, before 4 μl of assay media was added to each well to bring the final assay volume to 40 μl. The plate was incubated for 5 hours at 37°C/5% CO2 in a humidified incubator, and 8 μl of 1 μM substrate loading solution was added to each well before each plate was incubated for 2 hours at room temperature. Plates were read on a fluorescence plate reader, and each experiment was repeated three times.

Data analysis: Background-Subtracted Fluorescence (Fl = Fluorescence Intensity): Fl Sample − Fl Cell-Free Ctrl; Emission Ratio (using values corrected for background fluorescence): Coumarin Emission (460 nm)/Fluorescein Emission (530 nm); Response Ratio (Act. = Activation): Emission Ratio Compound/Emission RatioNo Act. Ctrl%InhibitionInhibitor Assays|{1Response RatioCompoundResponse ratioNo Act. CtrlResponse RatioEC80 CtrlResponse ratioNo Act. Ctrl}*100

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