Figure 7. Stat3 acts downstream of the insulin–InsR-mTOR signaling to up-regulate the expression of Ikaros. (A) Sorted MPPs infected with lentivirus encoding shRNAs or control shRNA (shCtrl) were cultured for 36 h, followed by analysis of Ikaros expression through RT-PCR. (B) Sorted MPPs cultured with or without insulin (insulin or CM, respectively) for 18 h were lysed for ChIP assays with anti-Stat3 antibody. Immunoprecipitates were examined with RT-PCR. DNA levels were normalized to input. (C and D) WT MPPs were transfected with empty vector or Stat3-vector for 24 h (C), followed by stimulation with 200 nM insulin for 18 h. Ikaros expression was analyzed through RT-PCR (D). (E) Stat3flox/flox mice were crossed with Vav-Cre mice to generate mice with conditional deletion of Stat3 in the hematopoietic system. Sorted MPPs were examined through immunoblotting. (F) Stat3+/+ or Stat3−/− MPPs were treated with 200 nM insulin for 18 h. Ikaros expression was examined through RT-PCR. (G) Sorted MPPs were cultured in StemPro-34 medium as described in Materials and methods. For mTOR activation (with insulin), insulin was added to the medium to a final concentration of 200 nM for 18 h. For mTOR inhibition (Rapamycin), rapamycin was added to the medium to reach 100 nM for 18 h. Phosphorylation of Stat3 was detected using antibodies against serine 727-phosphorylated Stat3 (p-Stat3 (S727)) or tyrosine 705-phosphorylated Stat3 (p-Stat3 (Y705)). (H and I) Stat3−/− MPPs were cotransfected with the indicated plasmids encoding WT Stat3, Y705F-Stat3, or S727A-Stat3 (H), followed by stimulation with 200 nM insulin or 100 nM rapamycin for 18 h. Ikaros expression was examined through RT-PCR (I). (J) Sorted MPPs were treated as in G and stained with antibody against Stat3, followed by confocal microscopy. Fluorescence intensity of nuclear Stat3 was calculated and shown as relative to total Stat3. (K) Stat3+/+ and Stat3−/− MPPs were seeded in differentiation medium for 10 d, and numbers of colonies were calculated. (L) 1000 sorted Stat3+/+ and Stat3−/− MPPs were seeded onto OP9 stromal cells and numbers of B220+CD19+ cells were calculated at the indicated intervals. (M) 5,000 sorted CD45.2 Stat3+/+ and Stat3−/− MPPs were 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. Bar, 2 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are from experiments repeated three times with similar results.
Figure 7.

Stat3 acts downstream of the insulin–InsR-mTOR signaling to up-regulate the expression of Ikaros. (A) Sorted MPPs infected with lentivirus encoding shRNAs or control shRNA (shCtrl) were cultured for 36 h, followed by analysis of Ikaros expression through RT-PCR. (B) Sorted MPPs cultured with or without insulin (insulin or CM, respectively) for 18 h were lysed for ChIP assays with anti-Stat3 antibody. Immunoprecipitates were examined with RT-PCR. DNA levels were normalized to input. (C and D) WT MPPs were transfected with empty vector or Stat3-vector for 24 h (C), followed by stimulation with 200 nM insulin for 18 h. Ikaros expression was analyzed through RT-PCR (D). (E) Stat3flox/flox mice were crossed with Vav-Cre mice to generate mice with conditional deletion of Stat3 in the hematopoietic system. Sorted MPPs were examined through immunoblotting. (F) Stat3+/+ or Stat3−/− MPPs were treated with 200 nM insulin for 18 h. Ikaros expression was examined through RT-PCR. (G) Sorted MPPs were cultured in StemPro-34 medium as described in Materials and methods. For mTOR activation (with insulin), insulin was added to the medium to a final concentration of 200 nM for 18 h. For mTOR inhibition (Rapamycin), rapamycin was added to the medium to reach 100 nM for 18 h. Phosphorylation of Stat3 was detected using antibodies against serine 727-phosphorylated Stat3 (p-Stat3 (S727)) or tyrosine 705-phosphorylated Stat3 (p-Stat3 (Y705)). (H and I) Stat3−/− MPPs were cotransfected with the indicated plasmids encoding WT Stat3, Y705F-Stat3, or S727A-Stat3 (H), followed by stimulation with 200 nM insulin or 100 nM rapamycin for 18 h. Ikaros expression was examined through RT-PCR (I). (J) Sorted MPPs were treated as in G and stained with antibody against Stat3, followed by confocal microscopy. Fluorescence intensity of nuclear Stat3 was calculated and shown as relative to total Stat3. (K) Stat3+/+ and Stat3−/− MPPs were seeded in differentiation medium for 10 d, and numbers of colonies were calculated. (L) 1000 sorted Stat3+/+ and Stat3−/− MPPs were seeded onto OP9 stromal cells and numbers of B220+CD19+ cells were calculated at the indicated intervals. (M) 5,000 sorted CD45.2 Stat3+/+ and Stat3−/− MPPs were 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. Bar, 2 µm. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are from experiments repeated three times with similar results.

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