Fig. 4

GGTase Is Required for Wnt/ß-catenin Signaling

(A) Knockdown of the ß subunit of PGGT1B reduced Wnt3A-induced transcriptional activation of the ß-catenin/TCF complex. MDA-MB-231 cells were transfected with a mixture of four siRNAs for PGGT1B and the reporter plasmids. The cells were treated with 50 ng/ml Wnt3A for 18 hr. Cont., control.

(B) Reconstitution of PGGT1B with Flag-tagged PGGT1B reversed PGGT1B knockdown-mediated reduction in transcriptional activation of β-catenin/TCF. MDA-MB-231 cells were transfected with a siRNA targeting the 32 untranslated region (32 UTR) of pggt1b gene, an expression vector for Flag-tagged PGGT1B deleted the 32 UTR, and the reporter plasmids. The cells were treated with 50 ng/ml Wnt3A for 18 hr. Fold activation to the absence of Wnt3A is shown. Values are ± SEM (n = at least 3). PGGT1B expression levels were analyzed by Western blotting with antibodies against PGGT1B and actin.

(C) Knockdown of PGGT1B inhibited BIO-induced goosecoid expression in zebrafish embryos. Embryos were injected with antisense MOs that target translation (ATG MO) or splicing (SP MO) of the zebrafish pggt1b gene at the one-cell stage and treated with 30 µM BIO or DMSO for 8 min at 32- to 64-cell stage. At 50% epiboly, embryos were fixed and subjected to in situ hybridization with an antisense goosecoid probe. Embryos are animal pole view, dorsal up if it can be distinguished. Arrowheads indicate tips of the goosecoid expression area. The central angles of goosecoid expression area were measured and scored into four classes: 0° to 90°, 90° to 180°, 180° to 270°, and 270° to 360° (n = at least 56).