Figure 5

334 promotes cell death by impairing the integrity of the mitochondrial membrane potential (MMP). (a) Short-term ClpXP inhibition by 334 reduces the cellular ATP levels. Jurkat cells were treated with 334 (3 h) and the cell viability was assessed by CellTiter-Blue™ (CTB) reagent. At the same time-points cellular ATP levels were measured by CellTiterGlo™ (CTG) reagent and the relative effect was compared to the cell viability at the indicated concentrations (*P < 0.05, t-test, Holm-Sidak method, n = 3). (b) 334 treatment drives mitochondrial ROS production. Mitochondrial ROS levels were assessed by flow cytometry using the MitoSOX™ reagent and relative superoxide levels are shown as mean fluorescence intensity normalized to the untreated control. Cells were treated with 334 as indicated (6 h) and the positive control was treated with antimycin A (Ant A) (20 µM, 45 min) prior to measurements (*P < 0.05, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (c) 334 promotes protein carbonylation by ROS-mediated oxidation reactions. Western blot shows bands of carbonylated proteins after treatment of Jurkat cells with 334 at the indicated concentrations (6 h). H₂O₂ pretreatment was used as a positive control (1 mM, 1 h) (left panel). Protein carboxylation was quantified and normalized to the untreated control (right panel) (One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (d) Short-term ClpXP inhibition by 334 disrupts the mitochondrial membrane potential without inducing apoptosis. JC-1 dye aggregates indicate mitochondria with intact MMP and were quantified by flow cytometry upon 334 treatment (30 µM) for the indicated time. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control for the loss of MMP (left panel). Apoptosis was assessed upon 334 treatment (30 µM) for the indicated time. Percentage of apoptotic cells as assessed by flow cytometry are shown (***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (e) Schematic overview of the impact of 334 treatments on mitochondrial and cellular functions. See also Supplementary Fig. S5 online. (f) 334 reduces basal respiration and respiratory capacity of leukemia cells. The oxygen consumption rate (OCR) over time was assessed for Jurkat, K562 and CEM cells untreated or treated with 334 (10 µM and 30 µM) for 24 h via mitochondrial stress test on an Agilent Seahorse XF Analyzer. OCR measurements were performed untreated and after injection of oligomycin (2.5 µM), FCCP (0.5 µM for K562/2.0 µM for Jurkat and CEM) and Rotenone/Antimycin A (0.5 µM). Basal respiration, spare respiratory capacity and proton leak were assessed as described in Supplementary Fig. S6a online. The FCCP titration for all cell lines is shown in Supplementary Fig. S6b online (*P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3).