Figure 2.
Endogenous MOZ is critical for MOZ/MLL fusion-mediated induction of target gene expression. (A) Gene expression concentrations of the Hoxa9/Meis1 genes in MOZ-TIF2, MLL-AF9, MLL-AF10, and AML1-ETO AML cells. Colonies of Moz+/+, Moz+/−, or Moz−/− AML cells that were serially replated >3 times were harvested and analyzed for Hoxa9, Meis1, and β-actin expression by quantitative reverse transcriptase-PCR (qRT-PCR). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin (n = 4 to 8). Error bars represent mean ± SD. Expression levels were compared using the Student t test; ∗P < .05 and ∗∗∗P < .005. N.T., not tested. (B) Expression of the Hoxa9/Meis1 genes in Moz+/+, Moz+/−, or Moz−/− cells bearing MLL-AF9 or MLL-AF10. The experimental scheme is shown at the top. Moz+/+, Moz+/−, or Moz−/− FL lineage− HSPCs were transduced with MLL-AF9, MLL-AF10, or empty vector (Mock) and cultured in a liquid medium. GFP+ cells were then sorted, and the expression levels of Hoxa9, Meis1, and β-actin were analyzed by qRT-PCR (n = 3 to 4). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin. Error bars represent mean ± SD. Expression levels were compared using the Student t test; ∗P < .05 and ∗∗∗P < .005. (C) Colony formation of Moz+/+, Moz+/−, or Moz−/− KSLs/CMPs bearing MOZ-TIF2 or MLL-AF9. The experimental scheme is shown at the top. First, Moz+/+, Moz+/−, or Moz−/− KSLs/CMPs (1 × 104 cells) were transduced with MOZ-TIF2 or MLL-AF9 fusion genes and cultured in a liquid medium. Subsequently, GFP+ cells (5 × 104) were sorted and cultured in a methylcellulose medium. Colony numbers were counted every 4 to 5 days, and then 3 × 104 cells were subsequently serially replated 3 times. The mean numbers of colonies formed by MOZ-TIF2– or MLL-AF9–expressing Moz+/+, Moz+/−, and Moz−/− cells derived from KSL or CMP fractions were calculated from 3, 4, or 5 independent experiments. (D) Hoxa9 and Meis1 expression in MOZ-TIF2–expressing cells derived from KSLs or CMPs. Moz+/+, Moz+/−, or Moz−/− KSLs and CMPs were transduced with the MOZ-TIF2 or MLL-AF9 fusion genes and cultured in a liquid medium. Then GFP+ cells were sorted, and gene expression levels of Hoxa9, Meis1, and β-actin were analyzed by qRT-PCR (n = 3 to 5). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin. Error bars represent mean ± SD; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005.

Endogenous MOZ is critical for MOZ/MLL fusion-mediated induction of target gene expression. (A) Gene expression concentrations of the Hoxa9/Meis1 genes in MOZ-TIF2, MLL-AF9, MLL-AF10, and AML1-ETO AML cells. Colonies of Moz+/+, Moz+/−, or Moz−/− AML cells that were serially replated >3 times were harvested and analyzed for Hoxa9, Meis1, and β-actin expression by quantitative reverse transcriptase-PCR (qRT-PCR). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin (n = 4 to 8). Error bars represent mean ± SD. Expression levels were compared using the Student t test; ∗P < .05 and ∗∗∗P < .005. N.T., not tested. (B) Expression of the Hoxa9/Meis1 genes in Moz+/+, Moz+/−, or Moz−/− cells bearing MLL-AF9 or MLL-AF10. The experimental scheme is shown at the top. Moz+/+, Moz+/−, or Moz−/− FL lineage− HSPCs were transduced with MLL-AF9, MLL-AF10, or empty vector (Mock) and cultured in a liquid medium. GFP+ cells were then sorted, and the expression levels of Hoxa9, Meis1, and β-actin were analyzed by qRT-PCR (n = 3 to 4). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin. Error bars represent mean ± SD. Expression levels were compared using the Student t test; ∗P < .05 and ∗∗∗P < .005. (C) Colony formation of Moz+/+, Moz+/−, or Moz−/− KSLs/CMPs bearing MOZ-TIF2 or MLL-AF9. The experimental scheme is shown at the top. First, Moz+/+, Moz+/−, or Moz−/− KSLs/CMPs (1 × 104 cells) were transduced with MOZ-TIF2 or MLL-AF9 fusion genes and cultured in a liquid medium. Subsequently, GFP+ cells (5 × 104) were sorted and cultured in a methylcellulose medium. Colony numbers were counted every 4 to 5 days, and then 3 × 104 cells were subsequently serially replated 3 times. The mean numbers of colonies formed by MOZ-TIF2– or MLL-AF9–expressing Moz+/+, Moz+/−, and Moz−/− cells derived from KSL or CMP fractions were calculated from 3, 4, or 5 independent experiments. (D) Hoxa9 and Meis1 expression in MOZ-TIF2–expressing cells derived from KSLs or CMPs. Moz+/+, Moz+/−, or Moz−/− KSLs and CMPs were transduced with the MOZ-TIF2 or MLL-AF9 fusion genes and cultured in a liquid medium. Then GFP+ cells were sorted, and gene expression levels of Hoxa9, Meis1, and β-actin were analyzed by qRT-PCR (n = 3 to 5). Expression levels of Hoxa9 and Meis1 were normalized to those of β-actin. Error bars represent mean ± SD; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .005.

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