Fig 2: Nevadensin induces Hippo-ON via MST1/2- LATS1/2-YAP signaling activation. A The levels of Phospho-MST, MST, Phospho-LATS, LATS, Phospho-Merlin and Merlin in nevadensin-treated Hep3B and HepG2 cells were examined by Western blotting. B, C Quantitative analysis of the levels of Phospho-MST/MST, Phospho-LATS/LATS, Phospho-Merlin /Merlin in nevadensin-treated Hep3B and HepG2 cells. n = 3, *p < 0.05; **p < 0.01, compared to control group. D The levels of Phospho-Yap and yap in nevadensin-treated Hep3B and HepG2 cells were examined by Western blotting. E Quantitative analysis of the levels of Phospho-Yap/Yap in nevadensin-treated Hep3B and HepG2 cells. n = 3, *p < 0.05; **p < 0.01, compared to control group. F The levels of Yap in Hep3B and HepG2 cells after treatment with nevadensin (12.5 µM), MG132 (10 µM) or both for 24 h were examined by Western blotting. G Quantitative analysis of the levels of Yap in treated Hep3B and HepG2 cells. n = 3, *p < 0.05; **p < 0.01, compared to control group

Fig 3: Merlin activation cycle.(A) Model for PIP2 mediated Merlin dimerization with “closed” monomers transitioning to “open” monomers upon PIP2 binding the proceeding to dimerization. (B) Model for Merlin activation cycle. Inactive closed conformation associated with Angiomotin. PIP2 binding leading to open conformation, dimerization and binding to ASPP2/Lats1/YAP1. Active complexes are phosphorylated leading to dissolution of the complex followed by dephosphorylation to regenerate the Merlin-Angiomotin complex.

Fig 4: Merlin dimerizes via the FERM domain.(A). A diagram depicting the dimerization assay using Merlin with either GFP or NanoLuc fused to its C-terminus. (B). A schematic diagram depicting the full-length Merlin (aa 1–595, WT) and the C-terminal deletion mutant (FH, aa 1–511), the FERM domain deletion mutant (HC, aa 315–595) and the dual FERM and CTD deletion (H, aa 315–511). (C). SDS-PAGE gels stained for total protein to access the quantity and purity of isolated probes for Merlin-NL (top) and GFP “bait” proteins (bottom) for GFP, Merlin-GFP, Merlin-FH-GFP, Merlin-HC-GFP and Merlin-H-GFP. (D). Merlin dimerization data quantified on a plate reader and presented as the bound luciferase normalized to 10% of the unbound input NL probe. The data is a mean of triplicate binding reactions with standard deviation and expressed as a percentage of the wild-type Merlin. Inset: an image of the light emitted from Merlin-NanoLuc bound to Merlin and Merlin deletion mutant-GFP bound magnetic beads, performed in triplicate, and dispensed into the wells of a 96-well plate (left). (E). A schematic diagram depicting the N- and C-terminal GFP fused “bait” and the N- and C-terminal fused Merlin-NanoLuc “Probe” constructs used in the BRET assays. (F). Triplicate Merlin dimerization assays for the N-and C-terminal Merlin constructs. The data is a ratio of bound to unbound NL probe, normalized to GFP fluorescence, (mean of triplicate binding reactions with standard deviation and expressed as a percentage of the peak value). (G). Emission spectrum from 400 nm to 600 nm of Merlin dimerization assays normalized to the 450 nm peak. The BRET emission peak at 510 nm is indicated by the arrow.

Fig 5: Merlin mutant dimerization.(A) A schematic diagram of Merlin mutants. Patient derived mutations: L64P, L360P and L535P, indicated by asterisks. ΔN18: deletion of the N-terminal 18 amino acids. N20: the first 20 amino acids fused to GFP. S518A/S518D: non-phosphorylatable and phosphomimetic mutants at S518. AR: closed conformation mutant. ΔEL: open conformation mutant. (B) Pulldown dimerization assays of patient derived Merlin mutants, L64P, Δ39–121, L360P, L535P and the lipid binding deficient mutant 6N. (C) Pulldown dimerization assays of Merlin N-terminal mutant, ΔN18, the unique N-terminal 20 amino acids fused directly to GFP, N20, the phosphorylation mutants S518A and S518D and the conformation mutants AR and ΔEL. GFP alone serves as the negative control. The data is a ratio of bound to unbound NL probe, normalized to GFP fluorescence, (mean of triplicate binding reactions with standard deviation and expressed as a percentage of the wild type Merlin. (D) Gel fractionation of lysates from cells transfected with plasmids expressing either wild type Merlin-NL (blue circles) Merlin-S518D-NL (red squares) or Merlin-S518A-NL (green triangles). (E) Gel fractionation of lysates from cell transfected with plasmids expressing either wild type Merlin-NL (blue circles) Merlin-AR-NL (red squares) or Merlin-ΔEL-NL (green triangles).