Figure 5. The IKAROS-PP1 interaction is required for normal hematopoietic differentiation.

Ikaros homozygote null (Ik^NULL) bone marrow-derived lineage negative (lin⁻) hematopoietic progenitor cells (HPCs) were transduced with pMSCV/Ik (Ik^NULL/Ik), pMSCV/IkΔPP1 (Ik^NULL/IkΔPP1) or the empty pMSCV vector (Ik^NULL/GFP); Ikaros wild type (Ik^WT) lin⁻ HPCs were transduced with the empty pMSCV vector only (Ik^WT/GFP); A) Scheme of the experimental procedure; Epo: Erythropoietin; ΔEpo: absence of Erythropoietin; B) Gene expression profiles in lin⁻ transduced HPCs; mRNA samples were retro-transcribed with oligo-dT nucleotides; cDNA was used as templates for qPCR with Ikaros, c-Kit, Flt3 or Gapdh specific primer sets; β-Actin was used as internal control; y axis: relative mRNA enrichment levels; ratios are plotted as the mean ± SD of the measurements; n = 3. *: P≤0.05 by Student's t-test; C) In vitro hematopoietic differentiation of lin⁻ HPCs. Clonogenic assays in methylcellulose of uninfected (Ik^WT lin⁻ and Ik^NULL lin⁻) or transduced (Ik^WT/GFP; Ik^NULL/GFP; Ik^NULL/Ik; Ik^NULL/IkΔPP1) lin⁻ HPCs; cells were seeded on methylcellulose and colonies were scored at day 14; CFU-GEMM: colony forming unit granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-G/M/GM: collectively identifies granulo-macrophage colonies, BFU-E: burst-forming unit erythrocyte; BFU-E/Mk: immature erythroid colonies with elevated megakaryocytic content; BFU-Mk/E: almost pure megakaryocyte colonies that contain only few clusters of immature erythroid cells; the data shown are representative of three independent experiments; table: absolute number of methylcellulose colonies; CFC: Colony Forming Cell; Ratio: ratio of methylcellulose colonies per number of plated lin⁻ HPCs; D–F) Cell proliferation and gene expression profiles in Ik^WT/GFP and Ik^NULL/GFP transduced lin⁻ HPCs upon SCF (Stem Cell Factor) and FL (Flt3 ligand) treatment; Ik^WT/GFP and Ik^NULL/GFP transduced lin⁻ HPCs were treated for 3 days with SCF and/or FL at the concentrations indicated at the bottom of the panels; D: cell proliferation and cell viability was determined by Trypan blue staining; y axis: cell number fold increase at day 0 and day 3; E, F: gene expression profile studies were carried out as detailed in panel B using β-Actin as internal control; y axis: mRNA enrichment levels relative to Ik^NULL-GFP/β-Actin values; the data shown in panels D–F are representative results of three independent experiments.