Torin1

Human osteosarcoma (OS) cell lines (HOS-MNNG and 143B) human rhabdomyosarcoma (RMS) cell lines (RD, alveolar RMS and Rh30, embryonal RMS) and the human breast cancer cell line MCF7 were obtained from the American Type Culture Collection (ATCC). Cells were grown in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin, L-glutamine and incubated at 37°C with 5% CO₂ and 100% humidity. FK-506 (tacrolimus, LC Laboratories), CCI-779 (temsirolimus, LC Laboratories), rapamycin (sirolimus, LC Laboratories), Torin1 (kind gift from Dr. Nathanael S. Gray, Dana-Farber Cancer Institute and David M. Sabatini, Whitehead Institute/MIT/HHMI), thapsigargin (#T-9033, Sigma-Aldrich Corporation) and AZD-8055 (kind gift from Astra Zeneca Corporation).

HeLa cells and HEK293T cells were purchased from ATCC. Hela TFEB-GFP cells were developed by the Shawn Ferguson lab^{23 (link)}. 621-102 and 621-103 cells were developed and characterized previously^{41 (link)}. All cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco/Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum with 1% penicillin/streptomycin. Rapamycin and Torin1 were purchased from LC Laboratories.

Cells were treated with drugs or starvation media Earl’s Balanced Salt Solution (EBSS) (Thermo Fisher, 24010043) 24 h after plating in 35 mm dishes or 96-well plates (50% confluence) for 24 h (unless specified) in an incubator at 37 °C with 5% CO₂. For astrocyte experiments, cells were treated 24 h after transfection under the same conditions described above. Drug treatments were at 1:1000 dilutions, using 0.1% DMSO as a vehicle control, and media removal and replacement as a control for starvation.
Drugs used were: rapamycin (LC—Laboratories, R-5000 or Tocris, 1292), Torin1 (LC—Laboratories, T7887), Bafilomycin A₁ (MilliporeSigma Calbiochem, 19600-010UG), non-commercial NAS were custom synthesized in house. We used NAS at 1 and 10 µM as having been shown effective in previous literature, including autophagy^{7 (link)} and GABA-A receptor modulation^{13 (link)}. Note that 10 µM of GABA-A receptor modulating NAS is supraphysiological and near the solubility limit for uncharged NAS.

Dasatinib, Torin 1, and rapamycin were obtained from LC Laboratories (Boston, MA). PP242 and RAD001 were obtained from Selleckchem (Houston, TX). PP2, U0126, and GSK 690693 were obtained from Sigma-Aldrich (St. Louis, MO). GSK 650394 was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). LY294002 was obtained from Cell Signaling (Danvers, MA). CGP 57380 was obtained from Abcam (Cambridge, MA).

To generate G1 arrested samples, 5 × 10⁵ RPE1‐hTERT cells were plated on a 10‐cm culture dish and treated with the following drugs: doxorubicin (100 ng/ml, Sigma‐Aldrich), palbociclib (5 μM, LC Laboratories), nutlin3 (10 μM, Cayman Chemical), and torin1 (1 μM, LC Laboratories). Cells were collected after 7 days of drug treatment.

Everolimus (LC Laboratories, E-4040), Torin 1 (LC Laboratories, T-7887), and silvestrol (MedChem Express, HY-13251) were reconstituted in DMSO and cells were treated at the concentrations indicated in the figures for 24 h. Phenformin (MedChem Express, HY-16397A) and eCF506^{32 (link)} (provided by Dr. Asier Unciti-Broceta) were reconstituted in sterile water, while rotenone (Toronto Research Chemicals, R700580), oligomycin A (Sigma, 75351), CCCP (Tocris, 04-525-00), GSK621 (Adooq, A15896), PF-06409577 (Tocris, 6114), and Dasatinib (LC Laboratories, D-330) were reconstituted in DMSO. Cells were treated at the concentrations indicated in the figures for 24 h. For treatment with GSK126 (Custom synthesis, Mercachem) and EPZ6438 (MedChem Express, HY-13803) (both reconstituted in DMSO), cells were treated at a concentration of 2 µM for 7 days, with media and drug replenished on day 3 and day 6. Cells were seeded into 96-well plates for use in proliferation assays or histones were extracted for immunoblotting analysis on day 7.