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. 2020 Jul 21;6:61. doi: 10.1038/s41420-020-00296-w

Fig. 3. MLN4924 activates the intrinsic apoptotic signaling pathway to elicit cell death.

Fig. 3

a Mitochondrial outer membrane potential (Δψm) was measured following a 48 h treatment with 30 nM or 100 nM MLN4924 via flow cytometry following TMRE staining (25 nM). b CellTiter-Glo® cell viability assay in control (EV) and HCT116 BAX/BAK double-knockout (DKO) cells treated for 72 h with MLN4924. Values were normalized to untreated control. IC50 values were calculated via a nonlinear regression model, based on the mean ± SD of three independent experiments. c Annexin-V/PI flow cytometry analysis of HCT116 EV and BAX/BAK DKO cells treated with 30 and 100 nM MLN4924 for 48 h. d Western blot analyses of PARP, BAK, BAX, CUL3, and β-actin expression in HCT116 EV and BAX/BAK DKO cells treated with 30 and 100 nM MLN4924 for 48 h. e Western blot analyses of DR5, Fas, BID, BIM, NOXA, PUMA, and β-actin expression following 24 and 48 h exposure of HCT116 p53+/+ and p53−/− cells to 30 and 100 nM MLN4924. f Western blot analyses of BID, PARP, caspase-3, DR5, procaspase-8, and β-actin following treatment of HCT116 EV BID CRISPR KO cells treated with 100 nM MLN4924 for 48 h. g High-content annexin-V/PI analysis of HCT116 EV and BID knockout (KO) cells following treatment with MLN4924 for 48 h. An unpaired Student’s T-test (Fig. 3a) or Two-way ANOVA with multiple comparisons test was used to compute p-values for mean ± SD of three independent experiments. Asterisks indicate statistically significant changes (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001; n.s denotes not significant).