Fig. 4

NR1 selectively inhibits mTORC1 but not mTORC2 in cells. a, b Effect of NR1 on insulin-dependent activation of mTORC1 and EGF-dependent activation of ^T202/Y204pERK1/2. MCF-7 cells were serum-starved for 16 h and treated with compound for 90 min before cell lysis and Western blot analysis. For insulin-stimulated cells, 100 nM insulin was added 30 min before cell lysis. For EGF-stimulated cells, 100 ng mL⁻¹ EGF was added 10 min before cell lysis. c Rap1 activity was detected in lysates loaded in vitro with either GDP or GTPγS and treated with NR1 at the indicated concentrations. This was followed by a subsequent pulldown of GST-RalGDS-RBD. Bound Rap1 was detected by Western blot. d Evaluation of NR1 in a LAM patient-derived cell line (TRI102) under replete and serum-starved conditions. For replete conditions, TRI102 cells were treated for 90 min with compound in DMEM + 10% FBS. For serum-starved conditions, TRI102 cells were serum-starved 16 h and then treated for 90 min with compound prior to cell lysis and Western blot analysis. e HEK293 cells co-transfected with the indicated mTOR constructs and HA-GST-S6K1 were starved of amino acids or treated with NR1. To assess mTORC1 activity, an HA IP was performed and Western blots probed for ^T389pS6K1. f Chronic treatment of PC3 cells with NR1 to evaluate the inhibition of mTORC1 (using ^T389pS6K1) versus mTORC2 (using ^S473pAKT). PC3 cells were treated with compound under replete conditions for 24 h prior to cell lysis and Western blot analysis. Rapamycin was used at 100 nM and Torin-1 at 250 nM