H4230 methylcellulose medium

De-identified primary AML patient specimens are from Toulouse University Hospital (TUH) (Toulouse, France). Frozen samples were obtained from patients diagnosed with AML at TUH after signed written informed consent for research use in accordance with the Declaration of Helsinki and stored at the HIMIP collection (BB-0033-00060). According to the French law, HIMIP biobank collection has been declared to the Ministry of Higher Education and Research (DC 2008-307, collection 1) and obtained a transfer agreement for research applications (AC 2008-129) after approbation by our institutional review board and ethics committee (Comité de Protection des Personnes Sud-Ouest et Outremer II). Clinical and biological annotations of the samples have been declared to the CNIL (Comité National Informatique et Libertés, i.e., Data Processing and Liberties National Committee). Patient characteristics are summarized in Figure S1A. Human primary AML cells were grown in H4230 methylcellulose medium (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 10% 5637-conditioned medium with or without ATRA (from 0.1 µM to 1 µM) and 1,25-Dihydroxyvitamin D3 (VD; from 10 nM to 100 nM) alone or in combination during 7 to 14 days at 37 °C with 5%CO₂.

Primary cells from AML patients were thawed and resuspended in 100 μl Nucleofector Kit V (Amaxa, Cologne, Germany). Then, cells were nucleofected according to the manufacturer’s instructions (program U-001 Amaxa, Cologne, Germany) with 200 nM siRNA scrambled (ON-TARGETplus Non-targeting siRNA #2, Dharmacon) or anti-CALCRL (SMARTpool ON-TARGETplus CALCRL siRNA, Dharmacon). Cells were adjusted to 1 × 10⁵ cells/ml final concentration in H4230 methylcellulose medium (STEMCELL Technologies) supplemented with 10% 5637-CM as a stimulant and then plated in 35-mm petri dishes in duplicate and allowed to grow for 7 days in a humidified CO₂ incubator (5% CO₂, 37 °C). At day 7, the leukemic colonies (>5 cells) were scored.

AML cells (10⁶/mL) were grown in duplicate in H4230 methylcellulose medium (Stem Cell Technologies) supplemented with 10% 5637-conditionned medium as described [50 (link)]. IRC-083864 was added at increasing concentrations in the culture medium. Cells were incubated for 7 days in a humidified CO₂ incubator. Leukemic colonies were then scored under an inverted microscope.