NOTCH activation correlates with Pten down-regulation and increased activation of PI3K/AKT pathway. (A) RNA from sorted wild-type LSK cells cocultured with OP9-GFP or OP9-DL1 for 3 days was used to perform analysis of Pten expression normalized to Gapdh and are shown relative to LSK cells grown on OP9-GFP. Mean ± SEM of triplicate experiments is represented. (B) Flow-sorted Lineage⁻ cells infected with either MSCV-IRES-GFP or MIG-ICN4 were fixed/permeabilized and prepared for phospho-flow analyses. Histograms show a representative of 3 independent animals from each group, and bar graphs indicate mean ± SEM. (C) Immunofluorescence on fresh frozen TNR BM sections probed with an anti-CD41 and an anti-GFP Ab and counterstained with DAPI shows that megakaryocytes are GFP⁺. (D) RNA was extracted from flow-sorted Lineage⁻cKit⁺GFP⁻ and Lineage⁻cKit⁺GFP⁺ cells of TNR animals, and quantitative RT-PCR analysis for PTEN expression was performed. Mean ± SEM of duplicate analysis is shown. (E) Flow-sorted Lineage⁻ cKit⁺ cells from TNR animals were fixed/permeabilized and stained for phospho-AKT or phospho-RPS6. Phosphorylation levels in GFP⁺ (active NOTCH signaling) and GFP⁻ (inactive NOTCH signaling) cells were assessed by flow cytometry. Histograms show a representative of 6 independent animals tested, and bar graphs indicate mean ± SEM.