Figure 5
The effect of double expression of D171N and BMI1. (A-C) IL-3–dependent 32Dcl3 cells were stably transduced with pMYs.IP/IB, pMYs.IP/BMI1, D171N/pMYs.IB, or D171N/BMI1. Before the assay for proliferation and apoptosis, the transduced 32Dcl3 cells were subjected to drug selection with 1 μg/mL puromycin and 10 μg/mL blasticidin. (A) G-CSF–induced differentiation assay in indicated 32Dcl3 transfectants. Surface expression of CD11b after incubation for 6 days in the presence of 1 ng/mL IL-3 (red histograms) or 50 ng/mL G-CSF (blue histograms) was analyzed by flow cytometry. The result of control staining is shown as a filled histogram. Data are representative of 2 independent experiments. The cells cultured with G-CSF for 6 days were assessed by Giemsa staining. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×1000. (B) Growth curve of the transduced 32Dcl3 cells cultured in the presence of 1 ng/mL of IL-3. Data are expressed as mean ± SD of 3 independent experiments. (C) Annexin V positivity in the transduced 32Dcl3 cells cultured without IL-3. Data are expressed as mean ± SD of 3 independent experiments. (D-G) Human CD34+ cells were precultured for 3 to 4 days in expansion medium and transduced with both GFP-tagged D171N and DsRed-tagged BMI1. After 3 to 4 days, GFP+/DsRed+ cells were purified by sorting. The cells were cultured in methylcellulose or long-term culture medium. (D) Expression of BMI1 and RUNX1-D171N were confirmed by Western blotting using anti-Bmi1 and anti–FLAG M2 antibodies, respectively. Anti–β-actin antibody was used as control. (E) Double-transduced cells were analyzed by CFC replating assay. Data are expressed as mean ± SD from 4 independent experiments. (F) LTC-IC assay in bulk and limiting dilution was carried out. (G) Growth patterns of the transduced cells cultured in long-term culture medium displayed as proliferation fold. The error bars represent the SD from 4 independent experiments. The growth profiles of all cells with double transduction of GFP (empty or D171N) and DsRed (empty or BMI1) vectors are shown. (H) Cell cycle analysis and the expression pattern of surface markers as shown by a typical flow cytometry profile, and Wright-Giemsa–stained cytospins of D171N- and D171N/BMI1-transduced cells on day 42 as captured with a BX51 microscope and a DP12 camera (Olympus); original magnification ×1000. (I) INK4A/ARF (p16/p14) expression levels in D171N- and D171N/BMI1-transduced cells on day 42. Relative gene expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of INK4A/ARF to GAPDH expression.

The effect of double expression of D171N and BMI1. (A-C) IL-3–dependent 32Dcl3 cells were stably transduced with pMYs.IP/IB, pMYs.IP/BMI1, D171N/pMYs.IB, or D171N/BMI1. Before the assay for proliferation and apoptosis, the transduced 32Dcl3 cells were subjected to drug selection with 1 μg/mL puromycin and 10 μg/mL blasticidin. (A) G-CSF–induced differentiation assay in indicated 32Dcl3 transfectants. Surface expression of CD11b after incubation for 6 days in the presence of 1 ng/mL IL-3 (red histograms) or 50 ng/mL G-CSF (blue histograms) was analyzed by flow cytometry. The result of control staining is shown as a filled histogram. Data are representative of 2 independent experiments. The cells cultured with G-CSF for 6 days were assessed by Giemsa staining. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); objective lens, UplanFl (Olympus); original magnification ×1000. (B) Growth curve of the transduced 32Dcl3 cells cultured in the presence of 1 ng/mL of IL-3. Data are expressed as mean ± SD of 3 independent experiments. (C) Annexin V positivity in the transduced 32Dcl3 cells cultured without IL-3. Data are expressed as mean ± SD of 3 independent experiments. (D-G) Human CD34+ cells were precultured for 3 to 4 days in expansion medium and transduced with both GFP-tagged D171N and DsRed-tagged BMI1. After 3 to 4 days, GFP+/DsRed+ cells were purified by sorting. The cells were cultured in methylcellulose or long-term culture medium. (D) Expression of BMI1 and RUNX1-D171N were confirmed by Western blotting using anti-Bmi1 and anti–FLAG M2 antibodies, respectively. Anti–β-actin antibody was used as control. (E) Double-transduced cells were analyzed by CFC replating assay. Data are expressed as mean ± SD from 4 independent experiments. (F) LTC-IC assay in bulk and limiting dilution was carried out. (G) Growth patterns of the transduced cells cultured in long-term culture medium displayed as proliferation fold. The error bars represent the SD from 4 independent experiments. The growth profiles of all cells with double transduction of GFP (empty or D171N) and DsRed (empty or BMI1) vectors are shown. (H) Cell cycle analysis and the expression pattern of surface markers as shown by a typical flow cytometry profile, and Wright-Giemsa–stained cytospins of D171N- and D171N/BMI1-transduced cells on day 42 as captured with a BX51 microscope and a DP12 camera (Olympus); original magnification ×1000. (I) INK4A/ARF (p16/p14) expression levels in D171N- and D171N/BMI1-transduced cells on day 42. Relative gene expression was measured by qRT-PCR performed in triplicate and calculated as the ratio of INK4A/ARF to GAPDH expression.

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