Figure 2

334 treatment impairs viability and colony formation of various leukemia cell lines. (a) ClpX and ClpP protein is expressed in various leukemia cell lines. Western blot analysis was performed for ClpX and ClpP in HL-60, K562, Jurkat, and CEM cells. The quantification of ClpX and ClpP protein expression was normalized to K562 and the respective protein load (n = 3). (b) 334 has strongest impact on cell viability of K562 and Jurkat cells. Cell viability of K562 and Jurkat cells upon 334 treatment (72 h) was normalized to the DMSO treated control (n = 3). (c) 334 treatment decreases cell viability of various leukemia cell lines. Cell viability of HL-60 and CEM cells upon 334 treatment for 72 h was normalized to the DMSO treated control. (d) IC₅₀ determination of various ClpXP inhibitors. Table shows IC₅₀ values calculated by nonlinear regression analysis. Missing data was not assessed (n.a.) or not calculated (n.c.) due to an insufficient inhibitory effect (n = 3). (e) 334 impairs clonogenic growth of K562 and Jurkat cells. Colony count of K562 and Jurkat cells normalized to untreated control cells is shown (left panels). Representative images of colonies formed by Jurkat and K562 cells pretreated with 334 for 24 h and grown in the absence of 334 for further 7 days are shown (right panel) (*P < 0.05, **P < 0.01, ***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). (f) 334 induces apoptotic cell death in K562 and Jurkat cells. Cell death upon 334 treatment (48 h) was assessed by flow cytometry using Nicoletti assay (upper left panel). Western blot analysis of PARP and procaspase-3 protein expression together with the respective whole protein loading controls are shown for K562 and Jurkat cells treated with 334 (50 µM, 24 h) (n = 3). The apoptotic cell death was assessed for cells treated with 334 (50 µM, 48 h), the pan-caspase inhibitor Q-VD-OPh (20 µM, 30 min) or a combination of Q-VD-OPh pretreatment prior to 334 exposure (50 µM, 48 h) (lower left panel). Cell death of HL-60 cells treated with 334 for 48 h is shown (lower right panel) (*P < 0.05, ***P < 0.001, One-way ANOVA, Tukey’s Multiple Comparison Test, n = 3). For (a) and (f) full length blots are presented in Supplementary Fig. S8 online. See also Supplementary Figs. S2 and S3 online.