INS synergized with active Notch1 (A-C) and exerted transforming activity in CLPs (D-F) in vivo. (A-C) Lin⁻ cells were cotransfected with vectors encoding the ICN1 gene (mock, ICN1) and the hIL7R gene (mock, WT, INS), followed by injection into lethally irradiated congenic mice. (A, upper) FACS analysis of the SP from WT/ICN1 or INS/ICN1 recipient mice at day 40. Data were obtained from GFP⁺ (marker for the ICN1 gene) and rat CD2⁺ (marker for the hIL7R gene) fractions. (A, lower) IHC of SP specimens using anti-CD3 antibodies from WT/ICN1 (left) and INS/ICN1 (right) recipient mice. Bar represents 20 μm. (B) Histological findings of liver (left 2 panels) and BM (right 2 panels) from WT/ICN1 (upper) and INS/ICN1 (lower) recipient mice (hematoxylin and eosin stain). Bar represents 50 μm. (C) Survival curves of recipient mice (mock/mock, n = 22; mock/ICN1, n = 44; WT/ICN1, n = 42; INS/ICN1, n = 54) **P < .01 (log-rank test). (D-F) CLPs transfected with hIL7R constructs were expanded in vitro for 18 days and injected into sublethally irradiated congenic mice. (D, top 2 panels) FACS analysis of the BM: WT recipient mice at day 60 (WT) and INS recipient mice at day 60 (INS). Data are obtained from GFP⁺ gated fractions. Open histogram, isotype control; shaded histogram, specific staining. (D, lower) Splenomegaly and lymphadenopathy developed in CLPs-INS recipient mice at day 60 (denoted as “INS”). (E) Survival curves of recipient mice (n = 11 for each condition). **P < .01 (log-rank test, INS vs WT or INS vs INS^2nd). (F) Histological findings: BM specimens from WT recipient mice (upper left) and INS recipient mice at day 60 by hematoxylin and eosin stain (lower left) or by IHC of B220⁺ cells (upper right). Lymph node cytospin from INS recipient mice at day 60 by May-Giemsa stain (lower right). Bar represents 20 μm. WT, WT primary recipients; INS, INS primary recipients; WT-2nd, WT day 30 BM secondary recipients; INS-2nd, INS day 30 BM secondary recipients.