Figure 5
Figure 5. MLL-ENL up-regulates Evi-1 expression exclusively in HSCs. (A) Defined hematopoietic populations were transduced with pMXs-neo-MLL-ENL or pMYs-HoxA9-ires-Meis1 and replated in semisolid medium. The expression level of Evi-1 (shaded bars; scale on the left) and HoxA9 (open bars; scale on the right) in MLL-ENL–transformed cells from each population (KSL MLL-ENL, CMP MLL-ENL, or GMP MLL-ENL lanes) and HoxA9/Meis1-transformed KSL cells (KSL HoxA9/Meis1 lane) was quantified relative to BM MNCs. Data are shown as mean ± SD from 2 independent experiments. *P < .05 vs CMP MLL-ENL, GMP MLL-ENL, or KSL HoxA9/Meis1, respectively. (B) KSL cells and GMPs were transduced with MIG (KSL/GMP GFP lanes), MIG-MLL-ENL (KSL/GMP MLL-ENL lanes), or MIG-MLL-AF9 (KSL MLL-AF9 lane). After 48 hours of transduction, the expression level of Evi-1 in GFP-positive cells was quantified relative to BM MNCs by real-time polymerase chain reaction and was compared with that of freshly isolated KSL cells (KSL lane), CMPs, and GMPs. Data shown are mean ± SD from 3 independent experiments. *P < .05. (C) BM progenitor cells from 5-fluorouracil–treated mice were transduced with MIG (GFP KSL lane), MIG-MLL-ENL (MLL-ENL KSL lane), or MIG-MLL-AF9 (MLL-AF9 KSL lane). After 36 hours of transduction, the expression level of Evi-1 in GFP-positive cells isolated from the KSL population was quantified relative to BM MNCs and compared with that of freshly isolated KSL cells (KSL lane). Data are mean ± SD from 3 independent experiments. *P < .05. (D) BM KSL cells, CMPs, and GMPs were isolated from Evi-1+ (open bars) and Evi-1+/− (shaded bars) mice and transformed by MLL-ENL in the same way as in the myeloid progenitor transformation assay. Bar graph shows mean colony numbers ± SD in the third round of serial replating from 2 independent experiments. *P < .05. (E) BM KSL cells, CMPs, and GMPs were isolated from Evi-1f/− mice. After they were transformed by MLL-ENL as in (D), they were transduced with either pGCDNsam-eGFP or pGCDNsam-eGFP-iCre. GFP-positive cells were isolated, and colony-forming activity after Evi-1 deletion was assessed in the next round of plating. Bar graph shows colony count ratio of iCre-GFP–transduced cells compared with GFP-transduced cells. Data are mean ± SD from 2 independent experiments. *P < .05.

MLL-ENL up-regulates Evi-1 expression exclusively in HSCs. (A) Defined hematopoietic populations were transduced with pMXs-neo-MLL-ENL or pMYs-HoxA9-ires-Meis1 and replated in semisolid medium. The expression level of Evi-1 (shaded bars; scale on the left) and HoxA9 (open bars; scale on the right) in MLL-ENL–transformed cells from each population (KSL MLL-ENL, CMP MLL-ENL, or GMP MLL-ENL lanes) and HoxA9/Meis1-transformed KSL cells (KSL HoxA9/Meis1 lane) was quantified relative to BM MNCs. Data are shown as mean ± SD from 2 independent experiments. *P < .05 vs CMP MLL-ENL, GMP MLL-ENL, or KSL HoxA9/Meis1, respectively. (B) KSL cells and GMPs were transduced with MIG (KSL/GMP GFP lanes), MIG-MLL-ENL (KSL/GMP MLL-ENL lanes), or MIG-MLL-AF9 (KSL MLL-AF9 lane). After 48 hours of transduction, the expression level of Evi-1 in GFP-positive cells was quantified relative to BM MNCs by real-time polymerase chain reaction and was compared with that of freshly isolated KSL cells (KSL lane), CMPs, and GMPs. Data shown are mean ± SD from 3 independent experiments. *P < .05. (C) BM progenitor cells from 5-fluorouracil–treated mice were transduced with MIG (GFP KSL lane), MIG-MLL-ENL (MLL-ENL KSL lane), or MIG-MLL-AF9 (MLL-AF9 KSL lane). After 36 hours of transduction, the expression level of Evi-1 in GFP-positive cells isolated from the KSL population was quantified relative to BM MNCs and compared with that of freshly isolated KSL cells (KSL lane). Data are mean ± SD from 3 independent experiments. *P < .05. (D) BM KSL cells, CMPs, and GMPs were isolated from Evi-1+ (open bars) and Evi-1+/− (shaded bars) mice and transformed by MLL-ENL in the same way as in the myeloid progenitor transformation assay. Bar graph shows mean colony numbers ± SD in the third round of serial replating from 2 independent experiments. *P < .05. (E) BM KSL cells, CMPs, and GMPs were isolated from Evi-1f/− mice. After they were transformed by MLL-ENL as in (D), they were transduced with either pGCDNsam-eGFP or pGCDNsam-eGFP-iCre. GFP-positive cells were isolated, and colony-forming activity after Evi-1 deletion was assessed in the next round of plating. Bar graph shows colony count ratio of iCre-GFP–transduced cells compared with GFP-transduced cells. Data are mean ± SD from 2 independent experiments. *P < .05.

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