Hcd34 apc

The acute myeloid leukemia cell lines KG1α and Kasumi were kindly provided by Prof. PT Liu (Wellcome Trust Sanger Institute, UK) and were cultured in (Iscove's Modified Dulbecco's Medium) IMDM (HyClone, Thermo Scientific, MA, USA) or RPMI-1640 (HyClone, Thermo Scientific, MA, USA) medium supplemented with 10% fetal bovine serum (FBS, Gibco, Life Technologies, NY, USA) and 1% penicillin/streptomycin, respectively. For cell line-sorting experiments, cells were stained with hCD34-APC (eBioscience, USA) and hCD38-PE (ebioscience, USA) for 30 min at 4 °C and were washed twice with PBS/1% FBS. Then the cells were sorted by flow cytometry (FACS Aria IIU, BD).

To assess apoptosis, CD34⁺CD38⁻ KG1α or Kasumi cells were cultured as described above for 24, 48, or 72 h with or without chidamide, Ara-C, DNR, or IDA, then double labeled with Annexin V-APC/PI (eBioscience, San Diego, CA, USA) for 15 min at room temperature in the dark according to the manufacturer’s instructions. Primary samples were stained with hCD34-APC (eBioscience, USA) and Annexin V-FITC/PI to assess the apoptosis of CD34⁺ primary cells induced by chidamide or IDA alone or the two drugs in combination. The stained cells were analyzed by flow cytometry (FACS Fortessa, BD Biosciences). Apoptotic cells were defined as Annexin V positive.

Human AML cell lines MOLM-13 and MV4-11 carrying MLL-rearrangement were purchased from ATCC (Rockefeller, MD, USA) and cultured at 37 °C in a 5% CO₂ incubator in Iscove’s modified Dulbecco’s medium (IMDM) and RPMI-1640 medium (HyClone, Thermo Scientific, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Grand Island, NY, USA), respectively. For sorting CD34⁺/CD38⁻ cells, cells were stained with hCD34-APC (eBioscience, Thermo Scientific, San Diego, CA, USA) and hCD38-PE (eBioscience, Thermo Scientific, San Diego, CA, USA) for 30 min at 4 °C. After washed twice with PBS containing 1% FBS, CD34⁺/CD38⁻ cells were sorted using FACS Aria IIU (Biosciences, Franklin Lakes, NJ, USA).