Figure 1. Insr knockout mice have more myeloid cells but fewer lymphoid cells. (A) Cells from BM were sorted through flow cytometry using the following markers: Lin− IL-7Rα− Sca-1+ c-Kit+ CD150+ Flk2− CD48− for LT-HSCs, Lin− IL-7Rα− Sca-1+ c-Kit+ CD150− Flk2− CD48− for ST-HSCs, Lin− IL-7Rα− Sca-1+ c-Kit+ CD150− Flk2+ for MPPs, Lin− IL-7Rα+ c-Kitlow Sca-1low for CLPs, Lin− IL-7Rα− Sca-1− c-Kit+CD34+ FcγRII/III− for common myeloid progenitors, Lin− IL-7Rα− Sca-1− c-Kit+ CD34− FcγRII/III− for MEPs, and Lin− IL-7Rα− Sca-1− c-Kit+ CD34+ FcγRII/III+ for GMPs. Insr was examined in sorted cell populations through RT-PCR. mRNA levels were first standardized to β-actin, and are shown relative to that of muscle cells. (B) The indicated cells were stained with anti-InsR antibody (green) and counterstained with propidium iodide (PI; red) for nuclei, followed by confocal microscopy. Fluorescence intensity of InsR was calculated and shown as relative to that of LT-HSCs. (C) InsR expression was examined in BM cells from Insr+/+ or Insr−/− mice using antibody against InsR-β. β-actin was probed as a loading control. (D) MxCre−;Insrflox/flox or MxCre+;Insrflox/flox mice were intraperitoneally injected with 300 µg poly(I:C) every other day for a total of three times. 8 wk after the last poly(I:C) injection, spleens were examined and the weight was shown in the right panel. (E) Hematoxylin and eosin staining of spleens 8 wk after the last poly(I:C) injection. (F) Leukocytes in spleens analyzed by flow cytometry at 8 wk after poly(I:C) administration. The following surface markers were used: CD11b for myeloid cells, CD11b and Gr1 for neutrophils, B220 for B cells, and CD3ε for T cells. For D–F, 7 Insr+/+ mice and 9 Insr−/− mice were used. (G) BM cells from Insr+/+ and Insr−/− mice were stained with CD11b for myeloid cells. (right) Percentages of CD11b-positive cells. (H) BM cells from Insr+/+ and Insr−/− mice were stained with IL-7Rα for lymphoid cells. (right) Percentages of IL-7Rα positive cells. n = 6 (G and H). (I and J) Kinetics of myeloid cells (I) and lymphoid cells (J) after poly(I:C) administration. n = 6 for each group. (K) Quantitation of BM subsets in Insr+/+ and Insr−/− mice at 4 or 8 wk after poly(I:C) injection. Surface markers were: B220 for B lymphocytes, CD11b+ Gr-1high F4/80− for granulocytes (Gran), CD11b+ Gr-1+ F4/80+ for monocytes (Mono). n = 7 for each group. (L) Numbers of lymphoid cells in peripheral blood of the indicated mice 16 wk after Insr deletion were examined through flow cytometry. Identifying markers were: CD3ε for T cells, B220 for B cells, and NK1.1 for NK cells. (M) Numbers of B cells and NK cells in BM of the indicated mice 16 wk after Insr deletion were calculated through flow cytometry. n = 5 (L and M). Bars: (B) 2 µm; (D) 100 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data presented above were repeated for three times with similar results.
Figure 1.

Insr knockout mice have more myeloid cells but fewer lymphoid cells. (A) Cells from BM were sorted through flow cytometry using the following markers: Lin− IL-7Rα− Sca-1+ c-Kit+ CD150+ Flk2− CD48− for LT-HSCs, Lin− IL-7Rα− Sca-1+ c-Kit+ CD150− Flk2− CD48− for ST-HSCs, Lin− IL-7Rα− Sca-1+ c-Kit+ CD150− Flk2+ for MPPs, Lin− IL-7Rα+ c-Kitlow Sca-1low for CLPs, Lin− IL-7Rα− Sca-1− c-Kit+CD34+ FcγRII/III− for common myeloid progenitors, Lin− IL-7Rα− Sca-1− c-Kit+ CD34− FcγRII/III− for MEPs, and Lin− IL-7Rα− Sca-1− c-Kit+ CD34+ FcγRII/III+ for GMPs. Insr was examined in sorted cell populations through RT-PCR. mRNA levels were first standardized to β-actin, and are shown relative to that of muscle cells. (B) The indicated cells were stained with anti-InsR antibody (green) and counterstained with propidium iodide (PI; red) for nuclei, followed by confocal microscopy. Fluorescence intensity of InsR was calculated and shown as relative to that of LT-HSCs. (C) InsR expression was examined in BM cells from Insr+/+ or Insr−/− mice using antibody against InsR-β. β-actin was probed as a loading control. (D) MxCre−;Insrflox/flox or MxCre+;Insrflox/flox mice were intraperitoneally injected with 300 µg poly(I:C) every other day for a total of three times. 8 wk after the last poly(I:C) injection, spleens were examined and the weight was shown in the right panel. (E) Hematoxylin and eosin staining of spleens 8 wk after the last poly(I:C) injection. (F) Leukocytes in spleens analyzed by flow cytometry at 8 wk after poly(I:C) administration. The following surface markers were used: CD11b for myeloid cells, CD11b and Gr1 for neutrophils, B220 for B cells, and CD3ε for T cells. For D–F, 7 Insr+/+ mice and 9 Insr−/− mice were used. (G) BM cells from Insr+/+ and Insr−/− mice were stained with CD11b for myeloid cells. (right) Percentages of CD11b-positive cells. (H) BM cells from Insr+/+ and Insr−/− mice were stained with IL-7Rα for lymphoid cells. (right) Percentages of IL-7Rα positive cells. n = 6 (G and H). (I and J) Kinetics of myeloid cells (I) and lymphoid cells (J) after poly(I:C) administration. n = 6 for each group. (K) Quantitation of BM subsets in Insr+/+ and Insr−/− mice at 4 or 8 wk after poly(I:C) injection. Surface markers were: B220 for B lymphocytes, CD11b+ Gr-1high F4/80− for granulocytes (Gran), CD11b+ Gr-1+ F4/80+ for monocytes (Mono). n = 7 for each group. (L) Numbers of lymphoid cells in peripheral blood of the indicated mice 16 wk after Insr deletion were examined through flow cytometry. Identifying markers were: CD3ε for T cells, B220 for B cells, and NK1.1 for NK cells. (M) Numbers of B cells and NK cells in BM of the indicated mice 16 wk after Insr deletion were calculated through flow cytometry. n = 5 (L and M). Bars: (B) 2 µm; (D) 100 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data presented above were repeated for three times with similar results.

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