Optimizing Pharmacological Inhibition Assays

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.