Fig. 1: The NEDDylation inhibitor MLN4924 enhances TRAIL-induced apoptosis.

a Schematic diagram of the Skp1-Cullin-1-F-box protein (SCF) Cullin-RING Ligase (CRL) complex and its regulation by NEDDylation. b High content microscopy analysis of cell death in HCT116 cells pre-treated with MLN4924 (100 nM) for 1 h, before treatment with rTRAIL for an additional 24 h. A student’s T test was performed between DMSO and MLN4924-treated samples (Error bars = SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = p > 0.05). c Western blot analyses and matched caspase-8 activity assays (d) in HCT116 parental and Bax-deficient cells co-treated with MLN4924 (100 nM) and rTRAIL (10 ng/mL) for 6 h. A two-way ANOVA was performed between Ctrl vs rTRAIL and MLN4924 vs Combo in each cell line (Error bars = SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = p > 0.05). e Western blot analyses of TRAIL-R2 DISC IP in HCT116 BAX-null cells pre-treated with MLN4924 (100 nM) for 1 h, prior to activation of the TRAIL-R2 receptor for the indicated times. f Western blot analysis of TRAIL-R2 DISC IP in HCT116 BAX-null cells treated for 1 h with MLN4924 (100 nM) or MG132 (10 µM) prior to activation of the TRAIL-R2 receptor for 1 h; cells were pre-treated with 20 µM zVAD-fmk pan-caspase inhibitor for 2 h as indicated.