Insulin–InsR signaling enhances Ikaros expression in MPPs through activation of mTOR. (A and B) Sorted MPPs infected with lentivirus encoding mTOR or control plasmid (Vector; A) were cultured for 24 h and analyzed by RT-PCR (B) after treatment with or without insulin for 18 h. CM, culture medium. (C and D) Sorted MPPs infected with lentivirus encoding shmTOR or control shRNA (shCtrl; C) were cultured for 36 h and analyzed by RT-PCR (D) after treatment with or without insulin for 18 h. (E) Sorted MPPs treated with rapamycin (100 nM) were cultured with or without insulin for 18 h, followed by RT-PCR. (F) WT MPPs were transfected with WT mTOR or kinase-dead mTOR (mTOR-KD) for 24 h, followed by stimulation with 200 nM insulin for 18 h. (G) Sorted MPPs pretreated as in E were seeded in differentiation medium for 10 d and numbers of colonies were calculated. (H) 1,000 sorted MPPs pretreated with or without 100 nM rapamycin were seeded onto OP9 stromal cells and numbers of B220⁺CD19⁺ cells were calculated at the indicated intervals. (I and J) MPPs were treated with 20 µM LY294002 for 12 h, followed by CFU assays (I) or OP9 co-culture (J). (K) 5,000 sorted CD45.2 MPPs were infected with lentivirus encoding Dox-inducible shCtrl or shmTOR sequences. After incubated with Dox for 36 h to induce the expression of shRNAs, cells with Dox washed off were then transplanted to normal or diabetic CD45.1 mice. 1 wk later, numbers of CD45.2 myeloid progenitors or lymphoid progenitors were calculated in BM by flow cytometry. n = 7 for each group. Data are shown as means ± SD. **, P < 0.01; ***, P < 0.001. Data are from experiments repeated three times with similar results.