Journal: Nutrients

Article Title: Polygonum cuspidatum Extract (Pc-Ex) Containing Emodin Suppresses Lung Cancer-Induced Cachexia by Suppressing TCF4/TWIST1 Complex-Induced PTHrP Expression

doi: 10.3390/nu14071508

Figure Lengend Snippet: Mammalian two-hybrid system-based high-throughput screening (HTS) for identification of natural compounds inhibiting TCF4–TWIST1. (Administrator) (A) Confirmation of interaction of TCF4 and TWIST1 by using a mammalian two-hybrid system. Principle of the mammalian two-hybrid system (upper panel). HEK293T cells were transiently transfected with GAL4-TWIST1, VP16-TCF4-full length (FL), VP16-TCF4-N-terminal domain (N), -middle domain (M) and C-terminal domain (C) expressing plasmids with pGL4.31-luciferase vector, and then cells were further incubated for 48 h. Luciferase activities were analyzed by using luciferase assay. ( B ) High-throughput screening for identification of natural compounds inhibiting the interaction of TCF4 and TWIST1. HEK293T cells were transfected with GAL4-TWIST1, VP16-TCF4-full length (FL) and pGL4.31-luciferase vector, and then transfected cells were incubated for 48 h prior to treatment of natural compounds library. Post-transfection, cells were incubated with 5 µg/mL of the natural compound for 24 h. Luciferase activities were analyzed by using luciferase assay. ( C ) Measurement of cell viability in hit compounds-treated cells. HEK293T cells were incubated for 24 h with each hit compound (5 µg/mL) as indicated. Cell viability was measured by crystal violet staining and assay. ( D ) Emodin is a potential small molecule targeting the interaction of TCF4 and TWIST1. HEK293T cells transfected with GAL4-TWIST1, VP16-TCF4-full length (FL) and pGL4.31-luciferase plasmids were incubated for 24 h with emodin as indicated, then luciferase activities were analyzed. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test.

Article Snippet: CheckMate TM /Flexi ® Vector Mammalian Two-Hybrid System Kit (Promega, Madison, WI, USA) was used to generate a mammalian two-hybrid luciferase assay system.

Techniques: High Throughput Screening Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Incubation, Staining