Figure 4. WNT5A overexpression is required for tumorigenic activity.

(A) Representative images of the colony formation assay for (iKras^– Yap1^Amp–) Wnt5a-KO cells. Clones without Wnt5a deletion were used as a control. (B) Quantification from triplicates is shown and is presented as relative colony numbers upon normalization to the shCtr group. (C) Western blot analysis for YAP1 and phospho-YAP1 in mouse PDAC cells treated with DMSO, 50 or 100 μM BOX-5. (D) Representative images of the colony formation assay in mouse PDAC cells of indicated genotypes treated with vehicle (DMSO) or BOX-5 (100 μM). (E) Quantification from triplicates is shown and is presented as relative colony numbers upon normalization to DMSO group. (F) Two independent clones of E-6 (iKras^– Yap1^Amp–) Wnt5a-KO cells and control cells without Wnt5a deletion (CTR) were subcutaneously injected into nude mice. Tumor volumes were measured on the indicated dates after injection. Results are presented as the means ± SD (n = 5). (G) E-4 (iKras^– Yap1^Amp–) Wnt5a-KO cells were infected with GFP, WNT5A, or YAP1^S127A and were subcutaneously injected into nude mice. Cells without Wnt5a deletion were used as a control. Tumor volumes were measured 30 days after injection. Results are presented as means ± SD (n = 5). (H) Subcutaneous xenograft tumors from G were stained for YAP1. Scale bar: 100 μm. (I) Percentage of cells with nuclear/cytoplasmic/double staining of YAP1. Error bars represent SD (n = 10 fields, 250 cells/field). P value was corrected with Dunnett’s method. (J) Representative images of the colony formation assay for PaTu8988T cells infected with WNT5A shRNAs or nontargeting shRNA (shCtr) (top). Quantification from triplicates is shown. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. Error bars from all panels indicate ± SD. *P < 0.05; **P < 0.01.