Cell Line Maintenance and Treatment Protocols

All cell lines were obtained from the American Type Culture Collection (Chicago, IL, USA), with the following exceptions: HCH-1 was from the laboratory of Dr. Linda Sandell at Washington University in St. Louis, MO, USA, and ASPS-1 was from the laboratory of Dr. Robert Shoemaker at the National Cancer Institute (Bethesda, MD, USA). SK-LMS-1, MNNG/HOS, MG-63, SK-UT-1 and SK-UT-1B cells were maintained in minimum essential media (Invitrogen, Waltham, MA, USA). Fuji, RD-ES, LUPI, NOS-1, HuO 9N2, HCH-1 and SK-ES cells were maintained in RPMI (Invitrogen). U-2 OS cells were maintained in McCoy's medium (Invitrogen). ASPS-1 cells were maintained in DMEM/F12 (Invitrogen). 293 T and SYO-1 cells were maintained in DMEM (Invitrogen). All media were supplemented with 10% fetal bovine serum (Invitrogen) and penicillin–streptomycin (Invitrogen). Chloroquine (used at 10 and 20 μM), 5-aza-dC (5 μM), necrostatin (10 μM) and ZVAD-FMK (100 μM) were obtained from Sigma Aldrich (St. Louis, MO, USA). Pepstatin A (used at 50 μM) and E64D (used at 25 μM) were obtained from Enzo Life Sciences (Farmingdale, NY, USA). ADI-PEG20 (used at 0.01, 0.05, 0.1, 0.5 and 1 μg/ml for dose-response curve, and at 1 μg/ml for all other in vitro experiments) was obtained from Polaris Pharmaceuticals, Inc. Lentiviral GFP-LC3 was a generous gift from Conrad Weihl (Washington University in St. Louis).^{27 (link)}