Figure 2. Insr-deficient LSKs tend to differentiate into myeloid cells but not lymphoid cells. (A) Representative flow cytometry patterns of BM cells prepared from Insr+/+ and Insr−/− mice. The indicated subpopulations were boxed. (B) Quantification of BM cells from Insr+/+ and Insr−/− mice. (A and B) Insr+/+, n = 6; Insr−/−, n = 5. (C) Insr+/+ and Insr−/− mice were intraperitoneally injected with BrdU (7.5 mg/kg) for 18 h, followed by analysis of BM GMPs and CLPs (left). Percentages of BrdU positive cells (right). (D) Cells from Insr+/+ and Insr-/- mice were first stained with Hoechst 33342, and then stained with pyronin Y, followed by flow cytometry (left). Percentages of cells in S/G2/M were calculated (right). (E) Insr+/+ and Insr−/− GMPs and CLPs were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (F) Long-term culture initiating cell (LT-CIC) assay of LSK cells from Insr+/+ and Insr−/− mice. (G) Equal numbers of Insr+/+ and Insr−/− cells were serially transplanted into lethally irradiated CD45.1 recipients. 16 wk later, repopulation of BM cells were analyzed by flow cytometry for numbers of CD45.2+ LSK cells. BMT, BM transplantation. (H) CFC (colony forming cell) assays of LSKs from BM of Insr+/+ and Insr−/− mice. Sorted LSKs were seeded in differentiation medium for 10 d and numbers of colonies were calculated. CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, macrophage. (I) Lymphoid differentiation potential of LSKs from BM of Insr+/+ and Insr−/− mice. 1,000 sorted LSKs were seeded onto OP9 stromal cells supplemented with cytokines needed for lymphoid differentiation and numbers of B220 and CD19 double-positive cells were calculated at the indicated intervals. (J) CFC assays of LSKs and MPPs from BM of Insr+/+ and Insr−/− mice. CFU-E, colony-forming unit erythroid. (C–J) n = 6. (K) Lethally irradiated CD45.1 mice were transplanted with BM cells from MxCre−;Insrflox/flox or MxCre+;Insrflox/flox mice. Reconstituted CD45.1 mice were then administrated with poly(I:C) and analyzed by flow cytometry 8 wk after Insr deletion. n = 7 per strain. (L) 100 Insr+/+ and Insr−/− LT-HSCs were cotransplanted with 3 × 105 CD45.1 BM cells into lethally irradiated CD45.1 mice, followed by progenitor cell examination in reconstituted BM 8 wk later. n = 8 per strain. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent at least three separate experiments.
Figure 2.

Insr-deficient LSKs tend to differentiate into myeloid cells but not lymphoid cells. (A) Representative flow cytometry patterns of BM cells prepared from Insr+/+ and Insr−/− mice. The indicated subpopulations were boxed. (B) Quantification of BM cells from Insr+/+ and Insr−/− mice. (A and B) Insr+/+, n = 6; Insr−/−, n = 5. (C) Insr+/+ and Insr−/− mice were intraperitoneally injected with BrdU (7.5 mg/kg) for 18 h, followed by analysis of BM GMPs and CLPs (left). Percentages of BrdU positive cells (right). (D) Cells from Insr+/+ and Insr-/- mice were first stained with Hoechst 33342, and then stained with pyronin Y, followed by flow cytometry (left). Percentages of cells in S/G2/M were calculated (right). (E) Insr+/+ and Insr−/− GMPs and CLPs were permeabilized and stained with antibody against active caspase-3. Cells positive for active caspase-3 were calculated. (F) Long-term culture initiating cell (LT-CIC) assay of LSK cells from Insr+/+ and Insr−/− mice. (G) Equal numbers of Insr+/+ and Insr−/− cells were serially transplanted into lethally irradiated CD45.1 recipients. 16 wk later, repopulation of BM cells were analyzed by flow cytometry for numbers of CD45.2+ LSK cells. BMT, BM transplantation. (H) CFC (colony forming cell) assays of LSKs from BM of Insr+/+ and Insr−/− mice. Sorted LSKs were seeded in differentiation medium for 10 d and numbers of colonies were calculated. CFU-GEMM: colony-forming unit-granulocyte, erythrocyte, monocyte/macrophage, megakaryocyte; CFU-GM: colony-forming unit-granulocyte, macrophage. (I) Lymphoid differentiation potential of LSKs from BM of Insr+/+ and Insr−/− mice. 1,000 sorted LSKs were seeded onto OP9 stromal cells supplemented with cytokines needed for lymphoid differentiation and numbers of B220 and CD19 double-positive cells were calculated at the indicated intervals. (J) CFC assays of LSKs and MPPs from BM of Insr+/+ and Insr−/− mice. CFU-E, colony-forming unit erythroid. (C–J) n = 6. (K) Lethally irradiated CD45.1 mice were transplanted with BM cells from MxCre−;Insrflox/flox or MxCre+;Insrflox/flox mice. Reconstituted CD45.1 mice were then administrated with poly(I:C) and analyzed by flow cytometry 8 wk after Insr deletion. n = 7 per strain. (L) 100 Insr+/+ and Insr−/− LT-HSCs were cotransplanted with 3 × 105 CD45.1 BM cells into lethally irradiated CD45.1 mice, followed by progenitor cell examination in reconstituted BM 8 wk later. n = 8 per strain. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data represent at least three separate experiments.

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