AZA induces proliferation of HSCs in vivo and reduces growth and viability of proliferating HSCs in vitro. (A) Treatment schema. C57BL/6 mice were injected intraperitoneally with AZA at 2.5 or 5 mg/kg per day for 3 days. After the last AZA dose, BrdU was administrated intraperitoneally at 1 mg per mouse once every 6 hours for a total of 24 hours. Mice were euthanized on day 4 after the start of experiment, and BM from both legs was analyzed by flow cytometry for incorporation of BrdU. (B) Representative histogram of BrdU expression in LT-HSCs (gated from live Lin^–Sca1⁺c-Kit⁺CD150⁺CD48^– cells) from 1 untreated and 1 AZA-treated mouse BM sample. (C) BrdU expression in percent for LT-HSCs (n = 4 mice per group). (D) Schematic of in vitro culture protocol. FACS-sorted mouse Lin^–Sca1⁺c-Kit⁺CD48^– or human Lin^–CD34⁺CD38^– cells were plated in 96-well flat-bottom plates coated with fibronectin that contained 100 µL of HSC media per well, and were supplemented with 10 ng/mL SCF and 100 ng/mL thrombopoietin (TPO). Different concentrations of AZA (0.1, 0.5, and 1 µg/mL) were added on 2 consecutive days (at baseline and at 24 hours after cell culturing). Cell counting and cell imaging was performed once every 6 hours for a total of 48 hours on an ImagExpress Pico automated cell counting system. After 48 hours, the percentage of live and dead cells was assessed by using EarlyTox Live/Dead Assay Kit. (E) Proliferation curves of FACS-sorted mouse Lin^–Sca1⁺c-Kit⁺CD48^– cells in the presence of indicated concentrations of AZA (left). Cell viability was assessed by the percentage of calcein AM⁺ cells after 48 hours of cell culture (right) (data are from 3 independent experiments). (F) Proliferation curves of FACS-sorted human Lin^–CD34⁺CD38^– cells in the presence of indicated concentrations of AZA (left). Cell viability was assessed by the percentage of calcein AM⁺ cells after 48 hours of cell culture (right) (data are from 2 independent experiments). Data are expressed as mean ± SD. *P < .05; **P < .01; ***P < .001.