![Figure 1. GATA-3 is abundantly expressed in LT-HSCs and plays a role in maintenance of the HSC pool. (A) qRT-PCR analysis of GATA-3 mRNA relative abundance in purified BM, thymus, and spleen populations recovered from adult (6- to 9-week-old) C57Bl/6 mice. LSK CD150+CD48−CD34− (LT-HSCs CD34−), LSK Flt3− (HSCs Flt3−), LSK Flt3lo (MPPs), LSK Flt3hi (LMPPs), Lin−Sca1loc-KitloIL-7Rα+Flt3+ (CLPs), Lin−c-KithiCD25− (ETPs), Lin−c-KithiCD25+ (DN2), Lin−c-Kitlo/−CD25+ (DN3), Lin−c-Kitlo/−CD25− (DN4), CD4+CD8+ (DP), CD4−CD8+ (thymic CD8 SP T cells), CD4+CD8− (thymic CD4 SP T cells), CD3+CD8+CD4− (splenic CD8 T cells), CD3+CD4+CD8− (splenic CD4 T cells), and CD19+ B220+ (splenic B cells). (−) indicates the no-template control. Data represent the summary of 3 independent experiments. (B) Loss of GATA-3 expression in LSK cells was confirmed through comparison of adult Gata3flox/flox:TgMx1Cre [f/f(+)] to control Gata3flox/flox [f/f(−)] or Gata3flox/+:TgMx1Cre [f/+(+)] 3 weeks after the first poly(I:C) injection. The level of GATA-3 mRNA of the f/f(−) mice was set at 100%. Data represent the summary of 5 mice of each genotype determined in 3 independent experiments with SEM. *P < .001. (C) Representative gating strategy of HSC populations in flow cytometric analysis. Lin− BM cells were isolated from f/f(−) and f/f(+) mice after TgMx1Cre induction by poly(I:C), and analyzed for surface expression of c-Kit and Sca1 to mark the LSK population, then further analyzed for CD150 and CD48 expression. Numbers adjacent to the boxed areas indicate the mean percentage of cells in the gate. (D) The absolute numbers of LSK CD150+CD48−CD34− cells per mouse (2 femurs plus 2 tibias) in f/f(−), f/+(+), and f/f(+) mice 3 weeks after poly(I:C) treatment. Data represent the summary of 12 mice of each genotype from 5 independent experiments with SEM. *P < .001; #P < .008. NS indicates not significant. (E) LSK CD150+CD48−CD34− cells were sorted from f/f(−), f/+(+), and f/f(+) mice 3 weeks after poly(I:C) treatment. Complete deletion of the conditional allele was verified in f/f(+) LT-HSCs. Data represent the summary of 3 mice of each genotype from 2 independent experiments with SEM. *P < .009; #P < .03. NS indicates not significant; ND, not detectable.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/119/10/10.1182_blood-2011-07-366070/4/zh89991287420001.jpeg?Expires=1723440198&Signature=QmbG4Erh4OMNdUvZLFSHVmbEkWyj~RcAabJ9dtzNOYHAdyr0f0k1uFP1gPqs2DxXbMk7JKXJoV001k6BBt2NLnt3J0qNpaxGSjYLTDTfSRxh-yaHVuQUN6gZ1gpn5MLgmqlnG75PLy16Y9EGwxguqSFoHVZbbwsn3wf92Dfta4gq~IObB1xtiVcNNb1xgKK6f7FMfKrr0eUEDHbJfl7aAkVxFH1B1Yx9WR5xLnHWsx5JtU7A7RdIewfJLmndgYXl6biHBdPUd4xHRnOeCr~0MssGyqUsZeEs8xfoDOYf7yXC~qH2KpxU-3N-TGWvlQ1AkweXv9LpFMe2zjll9AlIaw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
GATA-3 is abundantly expressed in LT-HSCs and plays a role in maintenance of the HSC pool. (A) qRT-PCR analysis of GATA-3 mRNA relative abundance in purified BM, thymus, and spleen populations recovered from adult (6- to 9-week-old) C57Bl/6 mice. LSK CD150+CD48−CD34− (LT-HSCs CD34−), LSK Flt3− (HSCs Flt3−), LSK Flt3lo (MPPs), LSK Flt3hi (LMPPs), Lin−Sca1loc-KitloIL-7Rα+Flt3+ (CLPs), Lin−c-KithiCD25− (ETPs), Lin−c-KithiCD25+ (DN2), Lin−c-Kitlo/−CD25+ (DN3), Lin−c-Kitlo/−CD25− (DN4), CD4+CD8+ (DP), CD4−CD8+ (thymic CD8 SP T cells), CD4+CD8− (thymic CD4 SP T cells), CD3+CD8+CD4− (splenic CD8 T cells), CD3+CD4+CD8− (splenic CD4 T cells), and CD19+ B220+ (splenic B cells). (−) indicates the no-template control. Data represent the summary of 3 independent experiments. (B) Loss of GATA-3 expression in LSK cells was confirmed through comparison of adult Gata3flox/flox:TgMx1Cre [f/f(+)] to control Gata3flox/flox [f/f(−)] or Gata3flox/+:TgMx1Cre [f/+(+)] 3 weeks after the first poly(I:C) injection. The level of GATA-3 mRNA of the f/f(−) mice was set at 100%. Data represent the summary of 5 mice of each genotype determined in 3 independent experiments with SEM. *P < .001. (C) Representative gating strategy of HSC populations in flow cytometric analysis. Lin− BM cells were isolated from f/f(−) and f/f(+) mice after TgMx1Cre induction by poly(I:C), and analyzed for surface expression of c-Kit and Sca1 to mark the LSK population, then further analyzed for CD150 and CD48 expression. Numbers adjacent to the boxed areas indicate the mean percentage of cells in the gate. (D) The absolute numbers of LSK CD150+CD48−CD34− cells per mouse (2 femurs plus 2 tibias) in f/f(−), f/+(+), and f/f(+) mice 3 weeks after poly(I:C) treatment. Data represent the summary of 12 mice of each genotype from 5 independent experiments with SEM. *P < .001; #P < .008. NS indicates not significant. (E) LSK CD150+CD48−CD34− cells were sorted from f/f(−), f/+(+), and f/f(+) mice 3 weeks after poly(I:C) treatment. Complete deletion of the conditional allele was verified in f/f(+) LT-HSCs. Data represent the summary of 3 mice of each genotype from 2 independent experiments with SEM. *P < .009; #P < .03. NS indicates not significant; ND, not detectable.
