Hl 60

Human AML cell lines U937, MV4-11, MOLM-14, HL-60 (DSMZ, Braunschweig, Germany) and KG1a (ATCC, Manassas, VA, USA) were maintained in minimum essential medium-α medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). All cell lines were grown in the presence of 100 units per ml of penicillin and 100 μg/ml of streptomycin, and were incubated at 37 °C with 5% CO₂. The cultured cells were split every 2–3 days and maintained in an exponential growth phase. All cell lines were annually re-ordered to DSMZ or ATCC stocks and periodically authenticated by morphologic inspection and mutational sequencing, and tested negative for Mycoplasma. The clinical and mutational features of our AML cell lines are described in Table 1.

Human AML cell lines HL60, U937 were purchased at DSMZ (Braunschweig, Germany) and KG1a at ATCC (Manassas, VA, USA). THP1 expressing IDH1^WT were gifted by Prof. Steven Chan [11 (link)]. Clones from the HL60 cell line expressing either IDH1^WT (clone 2 and 7) or IDH1^R132H (clone 5 and 11) were generated by our team [4 (link)]. HL60 was engineered to express IDH2^WT or IDH2^R172K as described below. All the cells excluding THP1 were maintained in MEMα with Glutamax (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen, Waltham, MA, USA) in the presence of 100 U/mL of penicillin and 100 µg/mL of streptomycin. THP1 were cultivated in RPMI with Glutamax (Gibco, Life Technologies, CA, USA) supplemented with 10% FBS (Invitrogen, MA, USA) in the presence of 100 U/mL of penicillin and 100 µg/mL of streptomycin. All cell lines have been routinely tested for mycoplasma contamination in the laboratory.

The THP-1 cell line is a monocytic leukemia cell line and was purchased from the German Collection of Microorganism and Cell Cultures (DSMZ, Braunschweig, Germany). The cell line was cultivated as recommended in RPMI 1640 with supplements and subcultivated every three to four days. The human myeloid leukemia cell line HL-60 was obtained from the DSMZ (Braunschweig, Germany). The cultivation was carried out in RPMI 1640 with supplements according to the specifications. For the ELISA both cell lines were plated at 2.4 x 10⁵ cells/well into 24-well plates, final volume 400 μL. The cells were incubated for 24 h at 37°C with 5% CO₂.
The 143B cell line is a human osteosarcoma cell line and was purchased from the American Tissue Culture Collection (ATCC, Wesel, Germany). The cells were maintained as recommended in DMEM with supplements. The Ewing’s sarcoma cell line TC-71 was obtained from DSMZ (Braunschweig, Germany) and was cultivated as recommended every two to three days in IMDM with supplements. For the ELISA the adherent cell lines were seeded at 1.2 x 10⁵ cells/well into 24-well plates and incubated as well for 24 h at 37°C with 5% CO₂.

The myeloid cell lines THP-1 and HL-60 were purchased from DSMZ (Braunschweig, Germany), authenticated and negatively tested for mycoplasma contamination. Cells were cultured in RPMI1640 medium (PAA laboratories, Cölbe, Germany) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (PAA laboratories). Stock solution (1 mM) of DAC dissolved in PBS was aliquoted and stored in −80 °C freezer and was diluted prior to each treatment (3x) in fresh RPMI medium to the required concentrations. Experiments were repeated three times.

Human myeloid leukemia cell lines (KG-1a, KG-1, HL-60, NB-4, ML-2, THP-1, MV-4-11, and U-937) were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Cells were cultured in RPMI medium (Life Technologies, Villebon-sur-Yvette, France) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2 mM L-glutamine (Life Technologies), 100 U/mL penicillin (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and 100 µg/mL streptomycin (Boehringer-Mannheim, Meylan, France) at 37 °C in fully humidified air and 5% CO₂. For all experiments, cells were harvested from culture while in their exponential growth phase.