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Fig. 3
GGTIs Specifically Inhibit the Wnt Canonical Pathway
(A) GGTI-286 rescued the eyeless phenotype in zebrafish embryos, but FTI-276, a farnesyltransferase inhibitor, did not. Embryos were pretreated with 100µm GGTI-286 or 100 µM FTI-276 at 50% epiboly, treated with 2µm BIO at the shield stage, and then incubated for another 24 hr. Images were taken at 30 hpf.
(B) GGTI-286 inhibited ß-catenin/TCF-dependent transcriptional activity, but FTI-276 did not. CHO cells were transiently transfected with the TOPFlash reporter plasmids. The cells were pretreated with 3, 10, or 30µm GGTI-286 or FTI-276 for 2 hr and treated with 2µm BIO for 18 hr. Firefly and Renilla luciferase activities were measured. Normalized relative luciferase activities are shown as fold activation to DMSO-treated cells.
(C and D) GGTI-286 (C) or GGTI-2174 (D) inhibited Wnt3A-induced transcriptional activation of the β-catenin/TCF complex. MDA-MB-231 cells were transiently transfected with the reporter plasmids. The cells were pretreated with 10µm GGTI-286 or 30µm GGTI-2174 for 6 hr and treated with 50 ng/ml Wnt3A for 18 hr. Firefly and Renilla luciferase activities were measured. Normalized relative luciferase activities are shown as fold activation to the absence of Wnt3A. Values are ± SEM (n = at least 3).
(E) GGTI-286 diminished expression levels of cyclin D1 and Axin2 in MDA-MB-231 cells. The cells were pretreated with 1 to 30µm GGTI-286 for 6 hr and treated with 50 ng/ml Wnt3A for 18 hr. Cell lysates were analyzed by Western blotting with antibodies against cyclin D1, Axin2, and actin.
(F) GGTI-286 reduced membrane localization of Rac1 in MDA-MB-231 cells. The cells were pretreated with 1 to 30µm GGTI-286 for 6 hr and treated with 50 ng/ml Wnt3A for 18 hr. Cell lysates were fractionated into the cytoplasmic and membrane fractions by ultracentrifuge and analyzed by Western blotting with antibodies against Rac1, Na, K-ATPase α1 subunit, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).