Murine NOTCH1 leukemias lacking Pten remain dependent on Notch signaling and are GSI sensitive. (A) Proliferation and (B) cell size analysis of primary mouse L1601P-ΔPEST leukemias on wild-type (WT) and Pten/Ink4a/Arf-null backgrounds treated with γ-secretase inhibitor (GSI) to block Notch signaling. Freshly explanted primary leukemia cells from different individual mice were cultured in vitro for 3 days with GSI (1μM compound E) or DMSO vehicle, pulsed with BrdU, and assayed by flow cytometry. Proliferation results are summarized from data plots presented in supplemental Figure 4. FSC indicates forward light scatter; ns, nonsignificant (Student t test). (C) Spontaneous loss of Pten protein expression occurred in one case (mouse nos. 3-7 as in Figure 1B) after 29 days of culture in vitro, yet it remained GSI sensitive. Pten protein expression was assessed by flow cytometry (right panel), and GSI response assayed by BrdU incorporation (left panel). Pten mRNA was detected by RT-PCR at both day 0 and day 29, and sequencing revealed no loss-of-function mutations (data not shown). Please note, only gated GFP⁺ events are depicted.