Journal: Blood Cancer Journal
Article Title: The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells
doi: 10.1038/bcj.2017.8
Figure Lengend Snippet: PTC596 reduces anti-apoptotic MCL-1 levels and induces mitochondrial apoptosis in AML. ( a ) BAX conformational changes, caspase-3 cleavage and Δψ m loss were determined by flow cytometry in MOLM-13 cells after 16-h (BAX activation) or 20 h (caspase-3 cleavage and Δψ m loss) exposure to 200 n M PTC596. The results are expressed as the mean±s.d. ( b ) Expression of BCL-2 family proteins in MOLM-13 cells after 20-h treatment with 50, 100 or 200 n M PTC596. The intensities of immunoblot signals were quantified and normalized to those of β-ACTIN. Levels in untreated cells were set at 1.0. White bars represent untreated controls. One-way ANOVA with Dunnett's test was used to determine significance between control and treated samples. NS, not significant. ( c ) MCL-1 expression in MOLM-13 cells treated with 200 n M PTC596 for indicated time periods. ( d ) MOLM-13 cells were preincubated for 1 h with 7 μM cycloheximide (CHX), 0.5 μM MG132 or 25 μ M Z-VAD-FMK, and MCL-1 levels were determined after 20-h treatment with 200 n M PTC596. β-ACTIN was used as loading control. Results are representative of three independent experiments.
Article Snippet: Antibodies against BMI-1, Histone H2A, Cyclin B1, CDK1, phosphorylated CDK1, phosphorylated CDK2, Securin and β-ACTIN were purchased from Cell Signaling Technology (Danvers, MA, USA); ubiquitinated histone H2A from EMD Millipore (Darmstadt, Germany); CDK2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); BCL-2 from Dako (Glostrup, Denmark); BCL-X L , MCL-1 and BAX from BD Biosciences; and conformation-specific BAX from Trevigen (Gaithersburg, MD, USA).
Techniques: Flow Cytometry, Activation Assay, Expressing, Western Blot
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