Culturing Cells with Myristoylated AKT1

Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a humidified 5% CO₂ atmosphere. TSC2-deficient ELT3V and TSC2-addback ELT3T cells are derived from Eker rat uterine leiomyoma, as described previously (46 (link)). TSC2-deficient and TSC2-addback cystadenoma 105K cell lines were obtained from Dr Elizabeth Henske’s laboratory (59 (link)). TSC2-deficient MEF infected with pBabe-Puro-Myr-Flag-AKT1, to generate TSC2-deficient MEF with myristoylated AKT1 (MEF-AKT1), and control TSC2-deficient MEF (MEF-EV) infected with EV were obtained from Dr Carmen Priolo’s laboratory (72 (link)). Myristoylation directs AKT to the membrane, keeping it constitutively phosphorylated at both Ser473 and Thr308 sites (72 (link), 73 (link)). Cells were serum starved overnight before treatment with indicated drugs/compounds, including losartan (100 nM (47 (link), 74 (link)); Tocris Bioscience), DMSO vehicle (Sigma-Aldrich), water (vehicle), or Klotho (100 ng/ml (55 , 56 ); R&D Systems) for indicated durations.