Fig 2: Hypoxia activates the MST1–FOXO1 cascade in primary cultured ECs. a GSEA analyses of the microarray data (GSE19284) obtained from isolated tip ECs and non-ECs by laser capture microdissection. b, c Immunoblot analyses and temporal changes of indicated proteins in HUVECs exposed to hypoxia (1% O2) for indicated times (n = 3, each group). Center line, median; Box limits, upper and lower quartiles; Whiskers, s.d. p values versus 0 h by one-way ANOVA with Tukey’s post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in HUVECs under normoxia (-) and hypoxia (+) in absence (-) or presence (+) of Trolox treatment. e Immunoblot analyses of indicated proteins in HUVECs under normoxia (-) and hypoxia (+) in absence (-) or presence (+) of Rotenone treatment. f Immunoprecipitation analysis in HUVECs with control anti-IgG and anti-FOXO1 antibody followed by immunoblotting with anti-MST1 antibody. g Immunoblot analyses of indicated proteins in siCont-ECs and siMST1-ECs under hypoxia. h Schematic picture depicting a hypoxia-intracellular ROS–MST1–FOXO1 cascade and the conserved phosphorylation site of FOXO1 by MST1. Source data are provided as a Source Data file

Fig 3: FGFR4 suppresses MST1/2 activation and cleavage in HER2+ breast cancer cells. a, b MDA-MB-453 cells transfected with indicated siRNAs were subjected to immunoblotting for a T183/180 phosphorylated MST1/2, and b MST1 and MST2. Note cleaved ~ 37 kDa MST1/N in FGFR4 knockdown cells (arrowhead). Thin gray line indicates cropping to leave out irrelevant sample lane; see uncropped immunoblots in Fig. S8. c MDA-MB-453 cells transduced with indicated shRNAs were transfected with siScr or siFGFR4 siRNA to 3’UTR before transfection of mock or FGFR4 (R) or (G) overexpression plasmid for a rescue experiment. Lysates were subjected to immunoblotting as indicated. Brackets indicate the cleaved MST1 and MST2 fragments. See Fig. S2A for phopsho-FRS2a and short exposure of MST1. d MDA-MB-453 and ZR-75.1 cells were transduced with indicated si/shRNAs; upper, indicated immunoblots of lysates; lower, quantification of pMOB1/MOB1 ratio, N = 3, mean ± SEM; *P < 0.05. For MST1/2 knockdown e ZR-75.1 and f MDA-MB-453 were transduced with shRNAs followed by transfection with siRNAs as indicated, and g BT474 cells were transfected with indicated siRNAs, and subjected to immunoblotting for pT183/180 MST1/2, MST1, MST2, and pMOB1 as indicated (in e arrowhead points to a full-length, bracket to the cleaved MST2) a–g. N = 3 independent repeats for all; except N = 2 in f and g

Fig 4: S100A1 regulates the YAP and Hippo signaling pathways. (A) S100A1 siRNA treatment upregulated p-LATS1 and p-YAP, and downregulated total YAP protein, but did not alter the expression of MST1/2 or p-MST1/2. (B) S100A1 siRNA treatment downregulated nuclear-YAP and upregulated cytoplasm-YAP protein. (C) Using CHX treatment, S100A1 depletion downregulated the half-life of YAP protein. (D) Co-immunoprecipitation analysis was performed in Hep3B cell lines. LATS1 co-immunoprecipitated with S100A1. The association was confirmed using a reciprocal approach. (E) Using the TEAD luciferase reporter, S100A1 siRNA downregulated the activity of TEAD in Hep3B and SMMC7721 cell lines. YAP, yes-associated protein; LATS1, large tumor suppressor kinase 1; MST, mammalian sterile 20-like kinase; siRNA, small interfering RNA; TEAD, TEA domain; CHX, cycloheximide; p-, phosphorylated.

Fig 5: FGFR4 substrate screen identifies tyrosine-phosphorylated Hippo pathway proteins including MST1/2. a Scheme of the substrate screen with recombinant FGFR4 kinase domain. b Top 10 FGFR4 substrates ranked by the Z-score include Hippo pathway -associated proteins (yellow). See Table S1 for the full substrate list. c, d MST1/2 are tyrosine phosphorylated by FGFR4 in COS-1 cells. Flag-tagged MST1/2 were immunoprecipitated after transfection of MST1 and MST2 alone or in combination with FGFR4 G388 (G), or R388 (R) kinase (wt), or kinase-dead (KD) variants, and detected by immunoblotting. e MST1 immunoprecipitates from COS-1 cells co-transfected with FGFR4 (R)-wt or FGFR4 (R)-KD (See Fig. S1A) were trypsin digested and subjected to phoshopeptide enrichment prior to LC-MS/MS analysis (N = 3) that identified phosphorylated Y433 (red) on MST1 only with FGFR4 (R)-wt, and phosphorylated S410 (green) only with FGFR4 (R)-KD