Figure 2. Effect of DACi for regulation of leukemia-initiating potential of AML1/ETO and PLZF/RARα positive stem cells. (A) Experimental strategy for studying the influence of DACi dacinostat and vorinostat on the biology of murine HSC. Sca1⁺/lin^- bone marrow (BM) cells were infected with the indicated retroviruses and maintained for one week in liquid culture supplemented with the indicated growth factors in the presence of DACi. All cells were inoculated into lethally irradiated recipients that were then sacrificed at day 12 after transplantation. (B) GFP reporter gene expression is given by FACS analysis of mock, AML1/ETO and PLZF/RARα infected Sca1⁺/lin^- cells. One out of three representative experiments is shown. (C) Proliferation of mock, AML1/ETO and PLZF/RARα expressing Sca1⁺/lin^- HSC exposed to DACi (20 nM dacinostat, 2 µM vorinostat). The cells were treated with indicated DACi for seven days and proliferation was determined by trypan blue exclusion of viable cells. Reported is the mean of fold change reduction of cell number with SEM compared with untreated controls in vitro (n = 3). (D) Numbers of spleen colonies are given as fold change reduction in CFU-S compared with untreated controls (n = 3). The colony numbers show the mean of three independent CFU-S12 experiments with SEM of mock, AML1/ETO and PLZF/RARα expressing Sca1⁺/lin^- HSC exposed to DACi (dacinostat, vorinostat) for seven days in vitro and transplanted into three mice per group. P-values: p = 0.02 (dacinostat-treated AML1/ETO expressing Sca1⁺/lin^- HSC), p = 0.005 (vorinostat-treated PLZF/RAR Sca1⁺/lin^- HSC) compared with mock-treated cells. (E) The expression of the leukemia-associated fusion proteins (LAFP) AML1/ETO and PLZF/RARα in the spleen colonies was assessed by RT-PCR. β-actin was used as a control. (n = 2) (F) For verification of the RT-PCR analyses, one quantitative real-time PCR analysis of the transgenes AML1/ETO and PLZF/RARα of the spleen colonies was performed. Internal reference gene was GAPDH. Results are represented as 2^-ΔΔCT values.