Figure 2. Yap1 is amplified in iKras⁻ relapse tumors and required for tumor growth.

(A) aCGH plots of relapse tumors shows that the 9qA1 locus containing Yap1 is focally amplified (denoted by red arrow) in E-1, E-2 and E-5. Normalized log₂ ratio for each probe are plotted. (B) IHC for Yap1 in relapse tumors. Note increased Yap1 expression in 9qA1 amplicon⁺ tumors, E-1, E-2 and E-5 but not 9qA1 amplicon⁻ tumor E-3. (C) qRT-PCR for Yap1 in relapse tumors. Relative expression levels normalized to reference gene. Error bars represent SD of the mean. (D) Representative wells of the clonogenic growth assay upon knockdown of Yap1 by two independent shRNAs primary cultures in Yap1 amplicon⁺ tumors (E-1 and E-2) and the iKras⁺ tumors (E-9 and E-10). Non-targeting shRNA (sh_Scr) was used as control. Quantification of cell growth is shown at the bottom. Error bars represent SD of triplicate wells, ***p < 0.001. (E) Xenograft tumor growth of cell cultures derived from E-2 expressing two independent Yap1 shRNA or control shRNA. Tumor volume was measured at the days indicated, data shown is representative of results from 2 independent experiments (n=5 per group). Error bars represent SD of the mean, **p < 0.01; ***p < 0.001. (F) IHC for proliferation marker Ki-67 and Yap1 in E-2 xenograft tumors expressing Yap1 shRNA described in (E) (G) Quantification of IHC staining for Ki-67 displayed as percentage of cells positive for Ki-67 staining. Error bars represent SD of the mean of 5 random fields, ***p<0.001).

See also Figure S2.