Figure 2
Figure 2. Deletion of Ezh2 compromises the proliferative capacity of MLL-AF9–transformed GMPs. (A) Growth of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed GMPs (1 × 104 cells each) with the indicated genotypes were cultured in IMDM with 20% FCS, SCF, FP6, GM-CSF, and IL-3 (10 ng/mL each) and their growth monitored every 3 days. The data are shown as the means ± SD for triplicate analyses. **P < .01. (B) Cell-cycle status and apoptosis of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed Ezh2+/+ and Ezh2Δ/Δ GMPs in panel A were pulsed with BrdU for 30 minutes and then stained with anti-BrdU Ab and 7-amino-actinomycin D. The percentage of cells in each phase of cell cycle and sub-G0/G1 apoptotic cells are shown as the means ± SD for triplicate analyses in a bar graph. *P < .05. (C) Replating efficiency of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed GMPs (1500 cells) with the indicated genotypes were serially replated in methylcellulose medium containing 50 ng/mL of SCF, 10 ng/mL of FP6, 10 ng/mL of GM-CSF, 10 ng/mL of IL-3, and 100nM 4-OHT. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. P1 and P2 denote platings 1 and 2, respectively. (D) Morphology of MLL-AF9–transformed GMP colonies. Representative colonies with indicated genotypes observed under an inverted microscope are depicted. Magnification ×100. (E) Typical cell morphology of MLL-AF9–transformed GMPs with indicated genotypes after 2 rounds of plating. Cells were cytospun onto glass slides and observed after May-Giemsa staining. Magnification ×400. (F) Effect of overexpression of Ezh2 in replating efficiency of MLL-AF9–transformed GMPs. MLL-AF9–transformed Cre-ERT;Ezh2+/+ GMPs were infected with a control vector virus or an Ezh2 lentivirus. Infected cells were purified by cell sorting using green fluorescent protein as a marker, and were serially replated as in panel C without 4-OHT. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. P1, P2, and P3 denote platings 1, 2, and 3, respectively.

Deletion of Ezh2 compromises the proliferative capacity of MLL-AF9–transformed GMPs. (A) Growth of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed GMPs (1 × 104 cells each) with the indicated genotypes were cultured in IMDM with 20% FCS, SCF, FP6, GM-CSF, and IL-3 (10 ng/mL each) and their growth monitored every 3 days. The data are shown as the means ± SD for triplicate analyses. **P < .01. (B) Cell-cycle status and apoptosis of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed Ezh2+/+ and Ezh2Δ/Δ GMPs in panel A were pulsed with BrdU for 30 minutes and then stained with anti-BrdU Ab and 7-amino-actinomycin D. The percentage of cells in each phase of cell cycle and sub-G0/G1 apoptotic cells are shown as the means ± SD for triplicate analyses in a bar graph. *P < .05. (C) Replating efficiency of MLL-AF9–transformed GMPs after deletion of Ezh2. MLL-AF9–transformed GMPs (1500 cells) with the indicated genotypes were serially replated in methylcellulose medium containing 50 ng/mL of SCF, 10 ng/mL of FP6, 10 ng/mL of GM-CSF, 10 ng/mL of IL-3, and 100nM 4-OHT. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. P1 and P2 denote platings 1 and 2, respectively. (D) Morphology of MLL-AF9–transformed GMP colonies. Representative colonies with indicated genotypes observed under an inverted microscope are depicted. Magnification ×100. (E) Typical cell morphology of MLL-AF9–transformed GMPs with indicated genotypes after 2 rounds of plating. Cells were cytospun onto glass slides and observed after May-Giemsa staining. Magnification ×400. (F) Effect of overexpression of Ezh2 in replating efficiency of MLL-AF9–transformed GMPs. MLL-AF9–transformed Cre-ERT;Ezh2+/+ GMPs were infected with a control vector virus or an Ezh2 lentivirus. Infected cells were purified by cell sorting using green fluorescent protein as a marker, and were serially replated as in panel C without 4-OHT. The black and gray bars represent compact and dispersed colonies, respectively. The data are shown as the means ± SD for triplicate analyses. **P < .01. P1, P2, and P3 denote platings 1, 2, and 3, respectively.

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