Figure 5. Insulin–InsR signaling controls the differentiation of LSKs through modulation of Ikaros expression in MPPs. (A) The indicated genes were examined in sorted cell populations through RT-PCR. mRNA levels were standardized to β-actin. (B) Expression levels of lymphoid and myeloid-related genes obtained above were subjected to gene set enrichment analysis and the normalized enrichment scores (NES) were calculated. (C) Sorted cells were cultured in StemPro-34 medium as described in Materials and methods. mRNA levels were quantified through RT-PCR analysis and results were first standardized to β-actin and then shown relative to that of LT-HSCs of Insr+/+ mice. CM, culture medium. (D) Mice were supplied with water only from 8:00 a.m. for 48 h, followed by refeeding normally for another 48 h. mRNA levels were analyzed as in C. (E) Sorted MPPs from mice treated with or without starvation for 48 h were stained with anti-Ikaros antibody (green) and counterstained with PI (red) for nuclei. Fluorescence intensity of Ikaros was calculated and shown as relative to that of starvation treated cells. (F) Single cell RT-PCR analysis of Flk2−LSKs and MPPs treated with or without insulin. n = 6. (G–J) 5,000 MPPs from WT mice were infected with lentivirus encoding the indicated doxycycline (Dox)-inducible shRNAs (G and H) or overexpressing plasmids (I and J). After incubation with Dox for 36 h (G and H) or 18 h (I and J) to induce the expression of shRNAs or overexpression plasmids, cells with Dox washed off were transplanted to lethally irradiated CD45.1 mice together with 3 × 105 CD45.1 BM cells. 1 wk later, numbers of the indicated CD45.2-positive cells were calculated by flow cytometry. (K and L) Sorted MPPs were infected with lentivirus containing the indicated plasmids for 24 h (K) and then seeded in differentiation medium for 10 d and numbers of colonies were calculated (L). (M) 1,000 sorted MPPs transfected with or without Ikaros were seeded onto OP9 stromal cells and numbers of B220+CD19+ cells were calculated at the indicated intervals. Bar, 2 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three independent experiments.
Figure 5.

Insulin–InsR signaling controls the differentiation of LSKs through modulation of Ikaros expression in MPPs. (A) The indicated genes were examined in sorted cell populations through RT-PCR. mRNA levels were standardized to β-actin. (B) Expression levels of lymphoid and myeloid-related genes obtained above were subjected to gene set enrichment analysis and the normalized enrichment scores (NES) were calculated. (C) Sorted cells were cultured in StemPro-34 medium as described in Materials and methods. mRNA levels were quantified through RT-PCR analysis and results were first standardized to β-actin and then shown relative to that of LT-HSCs of Insr+/+ mice. CM, culture medium. (D) Mice were supplied with water only from 8:00 a.m. for 48 h, followed by refeeding normally for another 48 h. mRNA levels were analyzed as in C. (E) Sorted MPPs from mice treated with or without starvation for 48 h were stained with anti-Ikaros antibody (green) and counterstained with PI (red) for nuclei. Fluorescence intensity of Ikaros was calculated and shown as relative to that of starvation treated cells. (F) Single cell RT-PCR analysis of Flk2−LSKs and MPPs treated with or without insulin. n = 6. (G–J) 5,000 MPPs from WT mice were infected with lentivirus encoding the indicated doxycycline (Dox)-inducible shRNAs (G and H) or overexpressing plasmids (I and J). After incubation with Dox for 36 h (G and H) or 18 h (I and J) to induce the expression of shRNAs or overexpression plasmids, cells with Dox washed off were transplanted to lethally irradiated CD45.1 mice together with 3 × 105 CD45.1 BM cells. 1 wk later, numbers of the indicated CD45.2-positive cells were calculated by flow cytometry. (K and L) Sorted MPPs were infected with lentivirus containing the indicated plasmids for 24 h (K) and then seeded in differentiation medium for 10 d and numbers of colonies were calculated (L). (M) 1,000 sorted MPPs transfected with or without Ikaros were seeded onto OP9 stromal cells and numbers of B220+CD19+ cells were calculated at the indicated intervals. Bar, 2 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three independent experiments.

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