Fig 2: YAP interacts with the mTORC2 component Sin1 and is phosphorylated on serine 436. (A) Co-immunoprecipitation of endogenous Sin1 and YAP. U87 cell extracts were immunoprecipitated with either Sin1 or YAP antibodies and immunoprecipitates subjected to immunoblot analysis for the indicated proteins. Lane 1, beads, no antibody; lane 2, immunoprecipitation with an irrelevant antibody (control IgG), lane 3, input cell lysate; lane 4 indicated immunoprecipitate probed with antibodies for the indicated proteins. (B) An in vitro kinase assay was performed with Rictor-immunoprecipitated mTORC2 from U87 cell lysates in the presence of recombinant AKT, native YAP or mutant YAP-5A as indicated in the presence of γ32P-ATP. Reactions were separated by SDS-PAGE followed by autoradiography. As negative controls, immunoprecipitated Mnk1 kinase or Raptor-immunoprecipitated mTORC1 was also used in the presence of YAP1, neither of which displayed detectable phosphorylation. (C) An mTORC2 in vitro kinase assay was performed utilizing Rictor immunoprecipitates isolated from U87 cells and incubated with purified native or S436A mutated recombinant Flag-tagged YAP for the indicated time points. Reactions containing native YAP were performed in the absence or presence of PP242 as shown. Kinase reactions were subsequently immunoblotted using a phospho-specific antibody generated against phospho-Ser436-YAP, Flag or Rictor antibodies for detection. (D) Effects of insulin stimulation in U87 cells. Cells were stimulated with insulin (10 nM, 5 h), lysed and extracts immunoblotted for the indicated proteins. (E) U87 cells were treated with siRNAs targeting Rictor or non-targeting control (siCtrl) for 24 h and cell lysates analyzed by immunoblotting for the indicated proteins. (F) As in (E) except U87 cells were treated with siRNAs targeting Sin1 or non-targeting control (siCtrl).

Fig 3: mTORC1 regulates G2/M checkpoint recovery. a, d U2OS cells were collected at different time points after IR (7 Gy) for cell cycle analysis, and the percentages of G2/M cells are presented in d. b The depletion of mTOR in a–e was detected by western blotting. c, e–n Cells were treated with IR (7 Gy) and 2 μM paclitaxel following different siRNA transfection in c, e, f, j–l, different mTOR inhibitor treatments (20 nM rapamycin or 1 μM KU0063794 for 24 h before IR) in g, either Ad5-CMV-empty or Ad5-CMV-Cre virus particle infection in h and i, or different concentrations of amino acids (AA; normal medium with 100% AA, 28 h before IR) in m and n. Cells were then stained with propidium iodide and p-H3 for mitotic entry analysis . The numbers in representative flow cytometry plots indicate percentages of mitotic cells, which were defined as p-H3-positive cells with 4N DNA contents. Protein samples at different time points were collected for western blotting . Actin was an internal control. Mock: cells incubated with only the transfection reagent; si-ctrl, si-Raptor, si-Rictor, or si-mTOR: cells transfected with non-target control siRNA, raptor, rictor, or mTOR siRNA, respectively; ctrl: control; error bars: mean ± SEM in d, e, g, j and mean ± SD in h, m; n = 3 independent experiments; *p < 0.05, two-tailed, unpaired Student's t-tests

Fig 4: Cand2 regulates MEF2 transcriptional activity Luciferase‐based quantification of MEF2 activity after Cand2 KD in NRCMs. Analyzed by t‐test, n = 3 biological replicates.mRNA levels of MEF2‐dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups. PE treatment for 24 h (50 μM). Analyzed by one‐way ANOVA, n = 6 biological replicates.mRNA levels of MEF2‐dependent genes Nr4a1 and Xirp2 in NRCMs after Cand2 OE. Analyzed by t‐test, n = 7‐8 biological replicates per conditions.RT‐qPCR analysis of Xirp2 mRNA levels in LVs of WT and Cand2 KO mice after sham and TAC surgeries. Analyzed by one‐way ANOVA, n = 6‐10 biological replicates per mice group.Grk5 protein and mRNA levels in NRCMs as well as MEF2 luciferase activity after 24 h of Torin 1 treatment. Analyzed by t‐test, n = 4–6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6).Cand2 and Grk5 protein levels after TSC2 KD. Analyzed by Western blot as well as MEF2 luciferase activity after TSC2 knockdown. Analyzed by t‐test, n = 5–6 biological replicates per conditions.Cand2 and Grk5 protein levels and Raptor transcript level in NRCMs after Raptor KD analyzed by Western blot. S235/236rpS6—phosphorylated ribosomal S6 protein, downstream target of mTORC1. Analyzed by t‐test, n = 3 biological replicates.Quantifications of Cand2 and Grk5 protein levels in NRCMs depleted of Raptor. Analyzed by t‐test, n = 3 biological replicates per conditions. Grk5, Nppa, and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD. Analyzed by t‐test, n = 3 biological replicates per conditions.Raptor and Grk5 protein levels after Raptor KD and adenoviral Grk5 overexpression.Cell surface area measurement of NRCM for indicated groups. Analyzed by one‐way ANOVA. n = 3 independent experiments, n > 150 cells.RT‐qPCR analysis of MEF2 targets in NRCMs after Raptor KD and Grk5 overexpression. Analyzed by one‐way ANOVA. n = 6‐8 biological replicates per conditions. Data information: Error bars indicate means ± SEM; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure.

Fig 5: Residue R840 in LARP1 is required for RAPTOR binding. (A, B) HEK293T cells were transiently transfected with empty vector (EV), human FLAG-tagged wildtype LARP1 (1019aa isoform) or the R840E/Y883A double-mutant. Cells were stimulated with complete growth media for 3 h and then lysed in CHAPS-based extraction buffer in the presence of RNase A. Lysates were subjected to immunoprecipitation with anti-FLAG antibody and analysed by SDS-PAGE/Western blot using the indicated antibodies.