Figure 4

334 induces cellular stress and alters the mitochondrial proteostasis. (a) Short-term 334 treatment (24 h) increases unfolded protein binding and decreases DNA repair. (b) Further, 334 treatment upon 48 h indicates the induction of the mtUPR. For (a) and (b) volcano plot analysis of quantitative MS-proteomics by SILAC are shown. Light, medium, and heavy isotope-labeled K562 cells were treated with 334 (10 µM) for the indicated time and compared to DMSO-treated controls (n = 3). Significantly enriched (blue) or depleted (orange) proteins are highlighted. Cut-off criteria: P < 0.05; |log₂(334 [10 µM]/DMSO)|> 1. See also Supplementary Fig. S4a,b, Supplementary Tables S1 and S2 online. (c) 334 reduces Hsp60 mRNA expression and induces transcription of CHOP. mRNA levels of Hsp60, ClpP, and CHOP were assessed in 334-treated Jurkat cells after 24 h and normalized to untreated controls. Relative mRNA expression was assessed by normalization to mRNA of the housekeeping gene actin (*P < 0.05, ***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (d) 334 treatment impairs Hsp60 and ClpP protein expression. Western blot analysis shows Hsp60 and ClpP protein levels in Jurkat cells after 334 treatment for 24 h (n = 3). Full length blots are presented in Supplementary Fig. S8 online.