Mechanism of pretubulysin (PT)-mediated downregulation of Mcl-1. (a) Real-time PCR analysis revealed that treatment of T24 and MDA-MB-231 cells with PT has no significant impact on mRNA expression of Mcl-1. (b) Inhibition of the proteasome by MG-132 counteracts the PT -mediated decrease of Mcl-1 protein levels. (c) Transfection of MDA-MB-231 cells with specific small interfering RNAs (siRNAs) against Fbw7 results in enhanced protein expression of Mcl-1. (d) Phosphorylation of Mcl-1, p38 and JNK is increased in PT-treated MDA-MB-231 cells after 24 h compared with DMSO-treated cells. Total Mcl-1, p38, JNK and actin levels served as control. (e) Inhibition of p38 pathways by 10 μM of the specific p38 inhibitor SB203580 had no influence on Mcl-1 expression, whereas treatment with 25 μM of the JNK inhibitor SP600125 results in clear upregulation of Mcl-1 expression in MDA-MB-231 cells. Combined treatment of the cells with the JNK inhibitor and PT stabilizes the protein level of Mcl-1. (f) MDA-MB-231 and T24 cells were pretreated for 18 h with 10 μM SB203580 or 25 μM SP600125 following incubation with 10 nM PT for 48 h. Fluorescence-activated cell sorter (FACS) analysis demonstrated that PT-induced apoptosis is not affected by inhibition of p38, whereas blocking JNK pathways is sufficient to abrogate apoptosis in PT-treated cells. *P<0.05 (one-way analysis of variance (ANOVA), Dunnett's test). Ctrl, control