Fig. 4: Primary MLL5 transduced APL blasts (GFP⁺ cells) exhibited increased cell proliferation and granulocytic differentiation in vitro and in vivo.

A Representative images of May-Grünwald-Giemsa-stained cytospins of transduced primary APL blasts (pMEG, and pMEG-MLL5) treated 8 days with ATRA (1 µM) and DMSO control (vehicle, 0.01%). B Fluorescence-activated cell sorting (FACS) immunophenotype of CD11b staining of transduced APL blasts with MLL5 or empty vector and C percentage of CD11b⁺ cells in transduced primary blasts after 8 days of ATRA (1 μM) treatment as the standard stimulus for differentiation. Data were expressed as mean ± standard error of the mean. D Schematic representation of cell proliferation assay based on GFP expression in primary APL cells transduced with MLL5 and the empty vector control (pMEG). Graphic bars represent the number of GFP⁺ cells inside the bulk of transduced primary APL blasts during 14 days of culture. Data were expressed as mean ± standard error of the mean. Overview of the mouse xenograft for APL. E Schematic representation of the generation of the xenograft mouse model for APL engraftment using NSGS mice. Representative FACS phenotype from a primary murine bone marrow transplanted with human transduced APL blasts with the empty vector (F) or the MLL5 gene (G) at sacrifice. APL blasts and mature myeloid committed cells were analyzed by flow cytometry using markers against CD117, CD33 and CD11b as indicated (inside the population huCD45⁺ and GFP⁺). Scatter plots showing engraftment of donor human CD45⁺ cells (regardless the GFP expression), human CD45⁺GFP⁺ cells and human CD45⁺GFP⁺CD11b⁺ cells in bone marrow (H), I spleen (percentual of engraftment and spleen weight) and J liver of transplanted mice at sacrifice. Data were expressed as median values. Ex vivo analysis of transduced APL blasts reinforces in vitro findings. L Incubation of bone marrow sorted APL blasts cells (GFP⁺CD45⁺CD117⁺CD33⁺) from pMEG/MLL5 engrafted mice, with ATRA plus ATO (1 μM each) led to increased induction of apoptosis over the course of 72 h in empty vector cells. Percentage of CD11b⁺ cells in sorted blasts from murine BM isolated from engrafted pMEG and MLL5 mice, after 8 days of ATRA alone or in combination with ATO (1 μM each) treatment as the standard stimulus for differentiation. Data were expressed as mean ± standard error of the mean. M Summary results from APL xenograft murine model to study the role of MLL5 in APL. *P < 0.05. **P < 0.01. ***P < 0.001. NS indicates not significant.