Legends to Supplementary Figures

LEGENDS TO SUPPLEMENTARY FIGURES

FIGURE 1S. Immunoprecipitaciton and blotting for MLL-AF4 and FLT3. (A) MLL-AF4 protein detection in two independent pools of transduced CB CD34+ HSPCs and MLL-AF4-carrying MV4;11 cell line. (B) Representative immunoprecipitation for FLT3 (bottom panel) and phospho-FLT3 blot (top panel) comparing transduced CB CD34+ HSPCs with MLL-AF4+ SEM and MV4;11 cell lines.

FIGURE 2S. Multilineage and multiorgan human chimerism in the IT, CL, spleen, liver and PB showing migration of human cells from the IT. No differences in the graft composition between EV-, MLLAF4-, FLT3.WT- and MLLAF4+FLT3.WT-expressing CD34+ HSPCs were found (n=46).

FIGURE 3S. FLT3.WT does not augment in vitro clonogenic potential but induces a transient in vitro expansion of MLL-AF4-expressing CD34+ HSPCs. (A) Primary and secondary CFU plating showing a higher CFU potential mediated by MLL-AF4 expression (n=4); E: erythroid, M: monocytic, G: granulocytic, GM: granulomonocytic, Mix: mixed. Inset: representative fluorescence-microscopy of CFU colonies transduced with a lentivector expressing dTo (EV or FLT3.WT), GFP (MLL-AF4) or merged-yellow (MLL-AF4+FLT3.WT). (B) In vitro cell expansion over a 90 days period of purified EV-, MLL-AF4-, FLT3.WT- and MLL-AF4+FLT3.WT-expressing CD34+ HSPCs (n=6 independent experiments from pooled CB units). Left insets: representative fluorescence images confirming reporter expression (GFP+, dTo+, merged-yellow) of FACS-purified transduced CD34+ cells. Right panels: representative RT-PCR and RT-qPCR confirming stable expression of MLL-AF4 and FLT3.WT, respectively, in long-term CD34+ cultures. (C) Apoptosis levels in CD34+ cells transduced with the indicated genes (n=3). (D) Proliferation level measured as % of BrdU+ cells within the CD34+ fraction transduced with the indicated genes (n=3). Please note that in vitro and in vivo experiments use different pools of CB-derived CD34+ cells.

FIGURE 4S. Co-cultures of transduced CB-CD34+ cells in OP9 stromal cells with hIL7 show that MLLAF4 (+/- FLT3 variants) does neither immortalize B-lineage cells nor alter B-cell differentiation potential. (A) Representative flow cytometry of normal B-lineage cells (CD19+CD10+) differentiated from transduced CD34+ cells. (B)MLL-AF4 (+/- FLT3) does neither immortalize B-lineage cells generated in vitro nor significantly alter B-cell differentiation potential.