Fig 2: The expression of PPARγ protein is dependent on MST2 and SAV1.(A) The expression of SAV1 and MST2 increased during the differentiation of 3T3-L1 cells. Expression of the indicated proteins and phosphorylation of MST2 were analyzed with antibodies in the lysates of 3T3-L1 cells prepared at various times after induction of differentiation. (B) The levels of endogenous PPARγ proteins were increased by overexpression of MST2 and/or SAV1 in 293 cells. (C) The levels of endogenous PPARγ proteins were increased by lentivirus-mediated overexpression of MST2 or SAV1 in 3T3-L1 adipocytes. (D) The levels of endogenous PPARγ proteins were decreased by shRNA-mediated knockdown of SAV1 expression in 3T3-L1 adipocytes. (E) The degradation of PPARγ protein was proteasome-dependent. The decrease of PPARγ protein induced by knock-down of SAV1 expression was inhibited by 5 µM lactacystin, a proteasome inhibitor.

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: A diagram represents the regulation of MST2 by IGF1/Akt signal.Under stress conditions, MST2 is activated through caspase-3 cleavage and autophosphorylation-Thr180. The activated/cleaved MST2 translocates to the nucleus and promotes apoptosis. IGF1/Akt inhibits these processes through phosphorylation of MST2-T117.

Fig 5: Depletion of Mst1/Mst2 affects proper EB differentiation.(A) Quantitative real time PCR to reveal the mRNA level of endoderm markers Gata6 and Sox17, mesoderm markers T and Gcs, and ectoderm markers Sox1 and Nestin in wild type EBs and Mst-/- EBs at day 4 and day 8 during EB formation. Actin was analyzed as an internal control. The data were shown as the mean ± S.D (n=3). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001). (B) Immunoblotting and densitometric analysis to check the protein level of Yap, YapS127, Oct4 and Nanog in day 4 and day 8 wild type EBs and Mst-/- EBs. Gapdh1 was analyzed as an internal control. The data are shown as the mean ± S.D (n=2). Statistically significant differences are indicated (*, P<0.05; **, P<0.01; ***, P<0.001).