Figure 2.

APC functions independently of β-catenin to restrict YAP activity. (A–C) β-Catenin, YAP, and p-YAP staining in serial sections of APC-deficient (Lgr5-EGFP-IRES-creER^T2;APC^flox/flox) jejunum 15 d after tamoxifen injection. Black arrows indicate APC-deficient crypts, and red arrows indicate normal crypts. Bar, 100 µm. (D–F) β-Catenin, YAP, and p-YAP staining in serial sections of β-catenin-stabilized (Lgr5-EGFP-IRES-creER^T2;Catnb^+/lox(ex3)) jejunum 15 d after tamoxifen injection. Black arrows indicate β-catenin-stabilized crypts, and red arrows indicate normal crypts. Bar, 100 µm. (G) Western blot analysis. Protein extracts from APC-deficient, β-catenin-stabilized, and control jejunal tissues 4 wk after tamoxifen injection were probed with the indicated antibodies. (Top gels) Equal amounts of the indicated extracts (1:1) were analyzed. (Bottom gels) Normal and diluted mutant extracts (1:0.1 for APC, and 1:0.5 for Δex3) were analyzed for YAP, p-YAP^S112, and p-YAP^S366. The ratios of p-YAP^S112 over total YAP and of p-YAP^S366 over total YAP were quantified. Data are mean ± SD. n = 3. (*) P < 0.05, t-test. (H) Western blot analysis. Protein extracts from APC-deficient and control jejunal tissues 4 wk after tamoxifen injection and protein extracts from normal tissues and adenomas of 3-mo-old APC^Min/+ mouse colons were probed with the indicated antibodies. (Top gels) Equal amounts of the indicated extracts (1:1) were analyzed. (Bottom gels) Normal and diluted mutant extracts (1:0.1 for APC mutant, and 1:0.2 for APC^Min/+ adenoma) were analyzed for Lats1 and p-Lats. (I) Western blot analysis. Protein extracts from HEK293 (control), HEK293 with CRISPR–Cas9-engineered APC knockout (APCKO), and HEK293 cells with APC or Lats1/2 RNAi were probed with the indicated antibodies. The ratios of p-Mst over Mst1, p-Lats over Lats1, p-YAP^S112 over total YAP, and p-YAP^S366 over total YAP were quantified. Data are mean ± SD. n = 3. (*) P < 0.001, t-test.