Dimethyl sulfoxide (dmso)

Primary hACs were cultured in 10 μl droplets (micromasses) in 24-well plates at a density of 300,000 cells/micromass. Culture medium was changed twice per week and consisted of DMEM/F12 (Gibco), 10% FBS (Gibco), 1% (vol/vol) antibiotic/antimycotic (Gibco), 1% L-glutamine (Gibco) and Insulin-Transferrin-Selenium (ITS) (Thermo Fisher Scientific). Micromasses were treated with vehicle (DMSO) or DOT1L inhibitor EPZ (10 μM) (Chemietek) under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 2 weeks. The micromasses were washed with PBS and fixed with ice-cold methanol for 1 hour at –20°C. After rinsing with PBS, the micromasses were stained with Alcian Blue (0.1% AB 8GX, MilliporeSigma) for 2.5 hours, washed with water, and air dried. Quantification of the staining was performed by dissolving the micromasses with 6 M guanidine (MilliporeSigma) for 6 hours and measuring the absorbance at 595 nm with a spectrophotometer (BioTek Synergy).

Dinaciclib (CAS 779353-01-4) was purchased from Selleckchem and diluted in DMSO (Sigma-Aldrich) to a 100 μM concentration. Used dilutions were made in complete medium. Pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk, CAS 187389-52-2) was purchased from ApexBio and diluted in DMSO to form a 50 mM stock solution. Used dilutions (40 μM) were made in complete medium. Generic BH3-mimetic ABT-737 (CAS 852808-04-9) was obtained from ApexBio and diluted in DMSO to an initial 10 mM stock. The Bcl-x_L specific inhibitor A-1331852 (CAS 1430844-80-6) was purchased from ChemieTek and a 3 mM initial stock solution in DMSO was employed. The nucleoside analog Gemcitabine (CAS 122111-03-9) was obtained as hydrochloride salt from LC Laboratories and diluted in DMSO to form a 1 mM stock. In every case, further dilutions were made taking into account not to surpass 0.1% (v/v) content of DMSO in media.