Figure 2: Triptolide restores multiple Wnt inhibitory factors expression to inhibit Wnt signaling.

a) Triptolide inhibits β-catenin/TCF-dependent transcription. TOPflash and FOPflash together with the pLG4.5 vector were co-transfected into A549 and H460 cells, and then treated with or without triptolide for 48 hours. The luciferase activity was normalized against Renilla activity and the activity of control cells transfected with TOPflash and FOPflash is designated as 100%. The experiments were performed in triplicate and representative data are shown. Results are expressed as mean±s.d. b) Inactivation of Wnt signaling in E160D mice. Immunoblot of total β-catenin, nuclear β-catenin and WIF1 protein in whole cell lysates, plus nuclear extracts from E160D mouse lung tissue. We compared seventeen-month-old wild type mice, E160D control mice, and E160D mice treated with triptolide. β-actin and H3 were used as the loading controls. c) Inactivation of Wnt signaling in A549 and H460 Xenograft mice. Immunoblot of total β-catenin, nuclear β-catenin, WIF1 protein in whole cell lysates, plus nuclear extracts of tumor tissue from mice treated with or without triptolide. β-actin and H3 were used as the loading controls. The actin loading control is the same as that in Figure 4b. d) A549 cells were stably transfected with shWIF1 or a scrambled shRNA control plasmid and then treated with 10 nM triptolide for 48 hours. Cell extracts were analyzed by immunoblot to determine the expression levels of WIF1, nuclear β-catenin, total β-catenin and nuprotein respectively. e) Colony formation assay of A549 lung cancer cells transfected with the shWIF1 or control plasmid cultured in the indicated concentrations of triptolide. f) A549 and H460 cells were treated with 2nM triptolide for 6 days and blotted against the indicated Wnt inhibitory factors using β-Actin as a loading control.