Protame

Eight E3 ligase inhibitors namely, Thalidomide, ProTAME, NSC 66811, Nutlin 3, HLI 373, JNJ 26854165, SMER 3 and NSC 146109 were used in the study. Thalidomide targets cereblon (CRBN), which makes an E3 ubiquitin ligase complex with damaged DNA-binding protein 1 (DDB1) and cullin-4A (Cul4A). Thalidomide binds with CRBN and inhibits the associated ubiquitin ligase activity [6 (link), 7 (link)]. ProTAME, a cell-permeable prodrug, converts to an active molecule, tosyl-L-arginine methyl ester (TAME) and inhibits the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C) [8 (link), 9 (link)]. NSC 66811 and Nutlin 3 have been reported as MDM2 (Murine Double Minute 2) E3 ligase inhibitors [10 (link), 11 (link)]. JNJ 26854165 and HLI 373 are HDM2 (Human Double Minute 2) E3 ligase antagonists. SMER 3 is an inhibitor of a yeast SCF family E3 ubiquitin ligase (SCFMet30). Thalidomide, NSC6811, NSC 146109 and SMER 3 were purchased from Tocris Bioscience. Nutlin 3, JNJ 26854165 and ProTAME were purchased from Selleckchem, USA, Axon Medchem, Netherland and Boston Biochem, Inc. USA respectively. Chloroquine (CQ) was used as the positive control, and dimethyl sulfoxide (DMSO) was used as vehicle control in this study. CQ and DMSO were obtained from Sigma-Aldrich, USA.

Ovsaho cells were obtained from JCRB (Japanese Collection of Research Bioresources Cell Bank) cell bank (NIBIOHN, Cell No. JCRB 1046) and grown in MCDB105/199 medium with 10% FBS (Fisher, #10438026) and substituted with 1% Penicillin-Streptomycin. All cells were free of Mycoplasma and their identity was verified by whole exome sequencing at the Broad Institute. For dose-response curves, cells were treated for 72 h with DMSO or the indicated doses of the PARP1 inhibitor AG-14361 (Selleckchem, #S2178), FAS/FASN inhibitor TVB-2640 (Selleckchem, #S9714), Bortezomib (Selleckchem, #S1013), proTAME (Selleckchem, #S9605), GC7 (Sigma Aldrich #259545). In combination assays, inhibitor concentrations were combined in a 1:1 ratio for indicated total concentration. Cell viability was measured using the software in a live-imaging IncuCyte station by counting live cells. Live cells were labeled by adding the Incucyte NucLight Rapid Red (NRR) Reagent (Essen Bioscience, #4717, 1:4000) to the medium. Datapoints of three biological experiments and three technical replicates (total n = 9) were merged and fitted using a non-linear log(inhibitor) versus normalized response with variable slope for dose-response curves using Prism (GraphPad). Combination Index (CI) was calculated based on Chou-Talaly method [53 (link)].