Fig. 3

The novel small molecule inhibitor EU-5346 specifically blocks Mcl-1 followed by cell death. a, b EU-5346 induces apoptosis in Mcl-1^wt/wt but not Mcl-1^Δ/null MEFs a. In contrast, ABT-199 induces apoptosis in both Mcl-1^wt/wt and Mcl-1^Δ/null MEFs b. MEFs were treated with indicated concentrations of EU-5346 a or ABT-199 b for 72 hours prior to Annexin V and PI staining. Data represent mean ± SD for triplicate samples. Results shown are representative of three independent experiments. c EU-5346-induced downregulation of Her2 and associated cell death are not triggered by caspase-mediated off-target effects of Mcl-1 siRNA. Her2-positive BC (SKBR3) cells were treated with EU-5346 and/or ZVAD (50 μM) and exposed to hypoxia during the last 6 hours. d EU-5346 inhibits proliferation of Her2-positive BC cells in a dose-dependent manner. BC cells were treated with EU-5346 for 3 days under hypoxic conditions. [³H]-thymidine was added during the last 8 hours. Data represent mean ± SD for triplicate samples. Results shown are representative of three independent experiments. c, d Whole-cell extracts were analyzed by immunoblotting with indicated antibodies. Immunoblotting for Erk2 confirmed equal protein loading. Erk2 extracellular signal-regulated kinase 2, Her2 human epidermal growth factor receptor 2, Hif hypoxia-inducible factor, kD kilodalton, Mcl-1 myeloid cell leukemia-1, MEF murine embryonic fibroblast, PARP Poly (ADP-ribose) polymerase