Myeloid immortalization of HSCs by MLL fusion genes associated with high Plzf expression. (A) Expression level of Plzf by QRT-PCR in myeloid immortalization assays. Tg LT-HSCs (CD34[⁻]), and ST-HSCs (CD34[⁺]) and MPs, retrovirally transduced with mock or CreER were harvested at the end of each round, and the first round, of plating, respectively. (B) Intracellular FACS analyses of Plzf expression in the immortalized cells constituting colonies of the CreER-transduced Tg LT-HSCs after the third round of plating. (C) Expression levels of Plzf by QRT-PCR in WT whole KSL or MP cells retrovirally transduced with mock, MLL-ENL, or MLL-SEPT6 at the first replating in myeloid immortalization assays. (D) Structure of Plzf (BTB/POZ domain and zinc fingers [Zn]) and a mutant (Plzf^ΔBTB) lacking the BTB/POZ domain, and expression of Plzf and the mutant by Western blot (lower left panels) and RT-PCR (lower right panels) analyses. Lysates extracted from Plat E cells transfected with mock (pMYs-IP), pMYs-Plzf-IP, or pMYs-Plzf^ΔBTB-IP were blotted with the anti-Plzf (αPlzf) antibody, followed by reprobe with the anti–α-tubulin antibody (αTub) as an internal control. The WT whole KSL and MP cells retrovirally transduced with mock, Plzf, or Plzf^ΔBTB were harvested at the first replating in myeloid immortalization assays and were subjected to RT-PCR. B2m was used as an internal standard. NTC, no template control. (E) Myeloid immortalization assays of WT LT-HSCs (CD34[⁻]), ST-HSCs (CD34[⁺]), whole KSL cells, and MPs retrovirally transduced with mock, Plzf, or Plzf^ΔBTB by replating 10⁴ cells. (F-H) Typical morphology of the colonies (F) in the Plzf-transduced KSL cells at the end of the third round of plating, and typical morphology (G) and immunophenotype (H) of the cells constituting these colonies. Colonies, and cells stained with Wright-Giemsa, were viewed with an Olympus CKX41 microscope using a 4×/0.13 objective lens and an Olympus BX41 microscope using a 20×/0.5 objective lens, respectively. Magnification and bars in panel F indicate 40× and 200 μm; magnification and bars in (G) indicate 200× and 20 μm. (I) Expression levels of Hoxa9, Meis1, and Evi1 in the immortalization assays, compared with the CreER-transduced Tg LT-HSCs (CD34[⁻]) by QRT-PCR. WT whole KSL and MP cells were retrovirally transduced with mock or Plzf and harvested at the end of the first and third rounds of plating. The CreER-transduced Tg LT-HSCs were harvested at the end of the third round of plating. The bar graphs show the mean ± SD of 3 independent experiments.