Figure 6
ClpP-KO impairs metabolic impact of 344 treatment. (a) Proliferation of HEK293T cells is impaired by knockout (KO) of the ClpXP complex. Cell number of HEK293T cells (HEK-WT) and two distinct ClpP-KO clones30 (HEK-KO1, HEK-KO2) was assessed by crystal violet staining 72 h after cell seeding and normalized to HEK-WT (***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (b) An intact ClpXP complex is required for 334-mediated reduction of ATP production. Viability with corresponding IC50 values (left panel) and ATP levels (right panel) of HEK-WT, HEK-KO1 and HEK-KO2 cells are shown. Cells were treated with 334 (10 µM, 30 µM, 50 µM, 100 µM) for 3 h (ATP production) or 72 h (Viability) and results were normalized to the untreated control of the respective cell type (*P < 0.05, **P < 0.01, paired t-test comparing HEK-KO1 or HEK-KO2 to HEK-WT, n = 3). (c) Respiratory capacity of ClpP-deficient (−/−) HEK (HEK-KO) cells is not altered by low-dose (10 µM) 334 treatment. The OCR over time was assessed for HEK-WT and HEK-KO1/HEK-KO2 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) 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 HEK-WT cells is shown in Supplementary Fig. S6b online (last panel) (*P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3).
