Apoptosis and Stemness Analysis

For apoptosis analysis, flow cytometric determination of annexin V binding, mitochondrial membrane potential loss (Δψ_m), conformational change in BAX and caspase-3 cleavage were carried out.^{29 (link)} For gating stem/progenitor population (CD34⁺CD38^low/− cells) in primary AML samples, anti-human CD34 and CD38 antibodies (BD Biosciences, San Jose, CA, USA) were used. Cell cycle distribution was analyzed by the Click-iT EdU incorporation kit (Life Technologies). Staining of intracellular BMI-1 and p53 was performed as described previously.^{19 (link), 30 (link)} Expression level was measured as the mean fluorescence intensity ratio (MFIR) calculated by the formula: MFIR=(MFI for specific antibody)/(MFI for isotype control).

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Nishida Y., Maeda A., Kim M.J., Cao L., Kubota Y., Ishizawa J., AlRawi A., Kato Y., Iwama A., Fujisawa M., Matsue K., Weetall M., Dumble M., Andreeff M., Davis T.W., Branstrom A., Kimura S, & Kojima K. (2017). The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells. Blood Cancer Journal, 7(2), e527-.

Publication 2017

Click-iT EdU incorporation kit

Annexin v Antibodies Antibody Apoptosis Caspase 3 Cell cycle Cells Cleavage Flow cytometric Fluorescence Human Intracellular Isotype Mitochondrial membrane potential Stem

Corresponding Organization :

Other organizations : Saga University, PTC Therapeutics (United States), The University of Texas MD Anderson Cancer Center, Chiba University, Kameda Medical Center, Bristol-Myers Squibb (United States)

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Variable analysis

independent variables

Annexin V binding
Mitochondrial membrane potential loss (Δψ_m)
Conformational change in BAX
Caspase-3 cleavage
Cell cycle distribution (EdU incorporation)

dependent variables

Apoptosis analysis

control variables

Gating stem/progenitor population (CD34+CD38low/- cells)
Staining of intracellular BMI-1 and p53
Expression level measured as mean fluorescence intensity ratio (MFIR)

controls

Isotype control for MFIR calculation

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