Extended Data Fig. 2: The Hippo pathway suppresses mTORC1 signalling depending on LATS1/2 kinase activity.
From: LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control
a,b, IB analysis of WCLs derived from HEK293A-WT, MAP4K4/6/7 (3KO) and MAP4K1/2/3/4/6/7 MST1/2 (8 KO) cells treated with 10 μM FSK (a) or 1 μg/ml LatB (b) for 60 min. c,d, IB analysis of WCLs derived from HEK293-WT and SAV1-KO cells at 30% (L) and 100% (H) confluence (c) or treated with 1 μg/ml LatB (d) for 60 min. e, IB analysis of WCLs derived from 293-WT and NF2-KO cells treated with 1 μg/ml LatB for 60 min. f, IB analysis of WCLs derived from 293 cells transfected with LATS1/MST1 or EV (empty vector, as a negative control). The cells were treated with 1 μM LPA or 1 μM S1P for 60 min before harvesting. g, LATS1/2 dKO cells were infected with EV, WT-LATS1 or kinase-dead LATS1 (AA) vector and selected with 200 μg/ml hygromycin for 72 hours to eliminate the non-infected cells. The resulting cells were cultured in 60 mm dishes at 30%, 60% and 100% confluence and harvested for IB analysis. h, In vitro kinase assays demonstrate that genetic deletion of LATS1/2 leads to increased mTORC1 kinase activity in vitro towards phosphorylating bacterially purified 4E-BP1. Raptor immunoprecipitates (IP) were prepared from HEK293A-WT or LATS1/2 dKO cells and used for examination of mTORC1 kinase activity in vitro. 250 nM mTOR inhibitor Torin 1 was added as indicated. Western blots in a–h were performed n = 2 independent experiments, with similar results obtained. Unprocessed immunoblots are shown in Source Data Extended Data Fig. 2.
