Fig 2: Alisol A attenuates YAP nuclear expression in NPC cells. (A) The docking interaction between alisol A and YAP was detected by AutoDock Vina software. (B) The docking interaction between alisol A and YAP was detected by Discovery Studio software. (C) The expression of Hippo signaling pathway-related proteins was detected by western blot. (D) YAP expression in cytosol or nuclear fractions was detected by western blot. (E) YAP expression in nuclear fractions was detected by immunofluorescence. Scale bar, 10 µm. Data are presented as the mean±SD of three independent experiments (*p<0.05, **p<0.01, ***p<0.001 vs. control group). NPC, nasopharyngeal carcinoma.

Fig 3: Yes inhibitors lower YAP1-mediated transcriptional network(A) Dose concentration treatment of dasatinib (μM) on the viability of UOK275, UOK342 and ACHN. HEK293 were used as controls. Cells were treated as indicated for 48 hours then viability was assessed by Cell-Titer Glo assay. (B) Yes-dependent phosphorylation of YAP1 and protein expression of survivin, cyclin D1 and CTGF was evaluated following 24 hours of dasatinib treatment by immunoblotting. (C) Expression of BIRC5 (survivin), CTGF (CTGF) and CCND1 (cyclin D1) was assessed by TaqMan assay 24 hours after dasatinib treatment (100nM) in HEK293, UOK275, UOK342 and ACHN.

Fig 4: Inhibition of the Hippo pathway is cytotoxic to NF2-deficient tumor cells(A) Basal protein expression level of YAP1 transcription targets survivin, cyclin D1 and CTGF were assessed by immunoblotting in UOK275, UOK342 and ACHN with HEK293 used to represent normal levels of expression. Β-actin expression levels were used as a loading control. (B) Gene expression of BIRC5 (survivin), CTGF (CTGF) and CCND1 (cyclin D1) in UOK275, UOK342 and ACHN. HEK293 were used as controls. (C) YAP1 nuclear localization in HEK293, UOK275, and UOK342 was quantified using an InCell Analyzer. (D) The effect of transient silencing of YES1 and YAP1 by small interference RNA or mock siRNA on cell viability was assessed in UOK275, UOK342, ACHN and HEK293 72 hours post-transfection by Cell-Titer Glo assay. N.S.: non-significant; siCTL: mock siRNA; siYES: siRNA against YES1; siYAP: siRNA against YAP1.

Fig 5: SKP2 interacts with YAP and induces its K63-linked polyubiquitination. a The HEK293T SFB-YAP stable cell line was transfected with MYC-tagged E3 ligases, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against FLAG and MYC. b The HEK293T SFB-YAP stable cell line was co-transfected with the K63-specific mutant of HA-ubiquitin and the indicated E3 ligase, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. c Co-immunoprecipitation of endogenous SKP2 with endogenous YAP. d In vitro binding of bacterially purified MBP-SKP2 to in vitro translated and purified GST-YAP. e In vitro binding of bacterially purified MBP-SKP2 to SFB-YAP purified from the HEK293T SFB-YAP stable cell line. MBP-SKP2 or MBP protein was incubated with SFB-YAP protein with or without CIP treatment at 37 °C for 1 h, followed by immunoblotting with antibodies against SKP2, p-YAP (S127), and FLAG. f The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48R or K63R) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. g The HEK293T SFB-YAP stable cell line was transfected with HA-ubiquitin (wild-type, K48-specific or K63-specific) and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. h Total K63-linkage specific ubiquitinated proteins in control or SKP2 knockout HEK293A cells (generated by CRISPR-Cas9) were immunoprecipitated by a K63-linked polyubiquitin-specific antibody, followed by immunoblotting with antibodies against YAP and ubiquitin. i siRNA targeting CUL1 or SKP1 was transfected into the HEK293T SFB-YAP stable cell line. Forty-eight hours after siRNA transfection, cells were transfected with HA-ubiquitin and MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. j The HEK293T UBCH5c shRNA stable cell line was transfected with SFB-YAP and HA-ubiquitin with or without MYC-SKP2, followed by pulldown with S-protein beads and immunoblotting with antibodies against HA and FLAG. k Purified GST-YAP protein was incubated with ATP, E1, UBCH5c, and the K63-specific mutant of His-ubiquitin with or without the SCFSKP2 complex, followed by immunoprecipitation with a YAP-specific antibody and immunoblotting with antibodies against ubiquitin and YAP