Figure 2.

Inactivation of the endogenous Hippo pathway recruits TICAMs. (A) Design of Cas9-mediated Lats1/2 knockout through HDI. (B) Lats1/2 knockout induces liver tumorigenesis. Representative livers 8 mo after injection, n = 5 mice. (C) HE and IHC staining of nontumor tissue and liver tumors induced by Lats1/2 knockout. Results are representative of three independent experiments. (D,E) The fragments of Lats1 (D) and Lats2 (E) genes targeted by sgRNAs were PCR-amplified from total tumor genomic DNA and sequenced. Representative sequences are shown. Red arrow lines denote predicted Cas9 cutting sites. (F) Tumors induced by Lats1/2 knockout exhibit a low YAP phosphorylation level as determined by Western blotting. Results are representative of two independent experiments. (G) Enhanced nuclear Yap level and CD45⁺ leukocyte recruitment to clones induced by Lats1/2 knockout. Immunofluorescence staining of post-injection day 30 serial liver sections for Yap and CD45. The arrow indicates an RFP-positive cell with nuclear Yap, and the arrowhead indicates an RFP-negative cell with cytoplasmic Yap. Results are representative of three independent experiments. (H) IHC staining of Lats1/2 knockout clones in post-injection day 30 serial liver sections. Note the oval cell-like morphology of cells with active Yap. Results are representative of three independent experiments. (I) Liver-specific Mst1/2 double knockout increases liver size. Livers at 2 mo of age were imaged. Images are representative of five mice. (J) Enhanced macrophage infiltration in Mst1/2 double-knockout livers. Sections of livers the same as those in I were stained for macrophage marker F4/80. Results are representative of three independent experiments.