HL-60 Cell Culture and Treatment

HL-60 human myeloblastic leukemia cells were a generous gift of Dr. Robert Gallagher. They are cultured in RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (GE Healthcare, Chicago, IL) and 1% antibiotic/antimycotic (Thermo Fisher Scientific, Waltham, MA) in a 5% CO₂ humidified atmosphere at 37  °C as previously described [34 (link)]. Retinoic acid (Sigma, St. Louis, MO) was added from a stock of 5 mM in ethanol to make a final concentration of 1 µM. Vacuolin-1 (Cayman Chemical, Ann Arbor, MI) was added from a stock of 0.25 mM in DMSO to make a final concentration of 0.25 µM. Viability assessed by 0.1% trypan blue exclusion was routinely in excess of 95% in all cultures except toxicity experiments in supplemental data that used vacuolin in excess of 2.5 µM where a sub-G1 population was also apparent.