Bix 02188

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

ERK5 inhibitors JWG-071 (Merck), AX-15836 (MedChemExpress), and MEK5 inhibitors BIX02188 and BIX02189 (Selleckchem) were diluted in dimethyl sulfoxide (DMSO, Sigma). Pan-caspase inhibitor Q-VD(OMe)-OPh (APExBIO) was diluted in DMSO. Death-receptor agonists, TRAIL (Peprotech) and TNFα (Merck) were diluted in H₂O 0.1% BSA, and anti-Fas activating antibody (Merck) was diluted in 50% glycerol in PBS.