Fig 2: Highly refractory B-cell acute lymphoblastic leukemic CD34+ cells expressing high levels of alkaline phosphatase activity (Case 1)Reference contour plots for five bone marrow (BM) aspirates and one peripheral blood (PB) analysis obtained in the last year of the follow-up, showing the performance of the alkaline phosphatase test in combination with CD34 staining. Prospective comparison and classification of ALP+ cells from new independent bone marrow samples corresponding to the same B-ALL patient, show different subsets of CD34+ cells. Each individual circle represents CD34+ cells with high expressing levels of alkaline phosphatase activity, presumably enriched in primitive refractory cells. Comparison of well-defined encircled populations consisting of CD34+/ALP high cells provides valuable information for dual parameter contour plots over time, to better discriminate subsets of apparently clonal CD34+ cells. First generation results data were obtained using blue laser excitation whereas NxT data were obtained using dual-laser excitation on the Attune Acoustic Focusing Cytometer and the Attune NxT Cytometer respectively (Thermo Fisher Scientific). Unlysed blood samples were simultaneously stained with PE-CD34, APLS, and DCV. The filter combination used consisted of 555 DLP, 530/30 (APLS), and 620 DLP, 574/26 (PE). For the Attune NxT, PE-CD34 was excited with the yellow laser at 561 nm and its emission was collected using the following filter combination: 595 LP, 600 DLP, and 585/16. APLS, PE and DCV fluorescence were displayed in logarithmic scale.

Fig 3: CG-745 increases helper T cells, cytotoxic T cells and natural killer T cells, and decreases Treg: (A) hPBMCs were incubated with CG (CG-745) for 36 hours and a subset of hPBMCs was analyzed using the antibodies indicated in the text; (B) hPBMCs were co-cultured with Huh7, Hep3B, HepG2 or PLC/PRF/5 cells for 36 hours with or without CG, and a subset of hPBMCs was analyzed by Attune Nxt (Invitrogen, USA).

Fig 4: CG-745 shows more potent HDAC inhibition compared to other HDACis and induces cell cycle arrest and cell death relatively early: (A) In vitro histone deacetylase inhibitory activity of CG (CG-745), Vor (vorinostat), Ent (entinostat), and Res (resminostat). HDAC activity was analyzed at different HDAC inhibitor concentrations by measuring HDAC substrate fluorescence. Results are shown as means based on experiments performed in triplicate (B) Cytotoxicity of each HDACis on Hep3B cells was analyzed with MTS following 72 hours post-exposure incubation (*p< 0.05); (C, D) Cells were stained by Annexin-V and PI (C), or PI only (D) following 24 hours post-exposure incubation, and samples were analyzed by Attune NxT (Invitrogen) for cell death and cell cycle; (E) Hep3B cells were incubated for 24 hours with HDACis and the total extract was analyzed for expression or phosphorylation of indicated proteins on a text by Western blot.