Gdc 0941

The small-molecule kinase inhibitors used included the PI3K catalytic inhibitors GDC-0941 (Cayman Chemical, 11600)^{44 (link)} and GDC-0032 (Selleck Chemicals, S7103)^{45 (link)} and BYL-719 (Active Biochem, A-1214)^{46 (link)}, the AKT catalytic inhibitor GDC-0068 (Selleck Chemicals, S2808)^{47 (link)}, and the AKT allosteric inhibitor MK-2206 (Cayman Chemical, 1032350-13-2)^{48 (link)}. Note that AKT catalytic inhibitors are known to increase AKT phosphorylation on threonine 308 and serine 473 by stabilizing a conformation in which these phosphorylated residues are inaccessible to phosphatases while, at the same time, fully inhibiting AKT kinase activity and phosphorylation of downstream substrates^{49 (link)}. Inhibitor stocks were dissolved in DMSO at 10 mM under sterile conditions, aliquoted and stored at −80 °C. Aliquots were freeze–thawed no more than five times. Insulin/growth factor treatments were preceded by 15 min of pretreatment with DMSO (Thermo Fisher Scientific, BP231-100) or a small-molecule inhibitor dissolved in DMSO. The inhibitors or DMSO were also present in the medium during labelling.

Vemurafenib (Selleckchem, S1267) was reconstituted at 4 mM in DMSO for the stock solution. Dabrafenib (Cayman, 16989-10) was reconstituted at 1.25mM in DMSO for the stock solution. Trametinib (Cayman, 16292-50) was reconstituted at 12.5 µM in DMSO for the stock solution. TGFB1 (R&D systems, 240-B-002) was reconstituted at 100 μg/mL in a 4mM hydrochloric acid, 1 mg/mL bovine serum albumin solution for the stock. PI3K inhibitor (GDC-0941, Cayman, 11600-10) was reconstituted at 10 mM in DMSO for the stock solution. TGFBRi (LY2109761, SML2051-5MG) was reconstituted at 40 mM in DMSO for the stock solution. IL6 (R&D systems, 206-IL-010) was reconstituted at 100 μg/mL in a 0.1% BSA PBS solution for the stock. EGF (R&D systems, 236-EG-200) was reconstituted at 200 μg/mL in PBS for the stock solution. BDNF (R&D systems, 206-IL-010) was reconstituted at 100 μg/mL in a 0.1% BSA PBS solution for the stock. EGF (R&D systems, 248-BDB-005) was reconstituted at 100 μg/mL in water for the stock solution.

Drugs used were Mitomycin C (M4287, Sigma, USA), MK-2066 (AdooQ), and GDC-0941 (Cayman), according to the manufacturer’s instructions, in a DMSO background which served as control. Information regarding concentrations is specified in the figure legends.

A549 cybrid cells (WT and mutant) and 143B cybrid cells were passaged every 3–4 days at 80% confluence and trypsinised using 0.25% trypsin-EDTA (Gibco #25200056). Similarly, control and patient fibroblasts were passaged every week in the same way. Media with or without drugs were changed every 2–3 days. For drug treatments, rapamycin (Cayman Chemical #13346), LY294002 (Cayman Chemical #70920) GDC0941 (Cayman Chemical #11600) and MK2206 (Cayman Chemical #11593) were dissolved in DMSO (Sigma-Aldrich #D2650) at 10 mM stock concentrations. Drugs were subsequently diluted to their working concentrations in media. Heteroplasmy level in the A549 cybrid cells, the 143B cybrid cells and the fibroblasts of patient 1 without treatments did not change significantly throughout the study as assessed by ARMS-qPCR or PCR-RFLP.