Figure 6.
Figure 6. Restoring PU.1 expression in PML-RARA leukemic cells induces granulocytic differentiation. (A) Western blot for PU.1 (top) detecting the PU.1-ER fusion protein in HT93A cells transduced with a PU.1-ER-expressing pBabePuro retrovirus (HT-PER) or with the vector alone (HT-V). Cells were treated with 1 μM β-estradiol as indicated. C indicates that Cos cells serve as a negative control and U937 cells as a positive control for endogenous PU.1 expression. The blot was also stained with an antibody against the M-CSF receptor (middle) and β-tubulin (bottom). (B) HT-PER cells and the control line HT-V were induced for 6 days with 1 μM β-estradiol. Fluorescence-activated cell sorting (FACS) analysis was performed at day 0 (left panels) and day 6 (right panels) using a PE-conjugated CD11b antibody (top panels) or a G-CSF receptor antibody (bottom panels). (C) HT-PER and HT-V cells were treated with 1 μM β-estradiol (Est) or 1 μM ATRA. Cell-cycle analysis was performed using propidium iodide staining at day 0 and 2. ▪ indicates the percentage of cells in G0-G1 stage, whereas □ indicates cells in G2-M stage and indicates cells in S phase. Median values are shown and error bars depict standard deviation from 3 independent experiments. (D) Wright-Giemsa staining of HT93A cells expressing the PU.1-ER fusion (HT-PER cells). The top 3 panels show unstimulated HT-PER cells (left panel), after 6 days of ATRA treatment indicating that the cells still differentiate toward neutrophils after having established the stable estrogen inducible system (middle panel), and after 6 days of treatment with 1 μM β-estradiol indicating differentiation toward neutrophils after 6 days (right panel). The bottom 3 panels depict the HT-V control line, which fails to show morphologic changes after β-estradiol treatment (right panel), but differentiates toward neutrophils after 6 days of ATRA treatment (middle panel). Images were visualized using a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) and a 100 ×/2 numeric aperture objective. Images were acquired using a Zeiss AxioCam camera.

Restoring PU.1 expression in PML-RARA leukemic cells induces granulocytic differentiation. (A) Western blot for PU.1 (top) detecting the PU.1-ER fusion protein in HT93A cells transduced with a PU.1-ER-expressing pBabePuro retrovirus (HT-PER) or with the vector alone (HT-V). Cells were treated with 1 μM β-estradiol as indicated. C indicates that Cos cells serve as a negative control and U937 cells as a positive control for endogenous PU.1 expression. The blot was also stained with an antibody against the M-CSF receptor (middle) and β-tubulin (bottom). (B) HT-PER cells and the control line HT-V were induced for 6 days with 1 μM β-estradiol. Fluorescence-activated cell sorting (FACS) analysis was performed at day 0 (left panels) and day 6 (right panels) using a PE-conjugated CD11b antibody (top panels) or a G-CSF receptor antibody (bottom panels). (C) HT-PER and HT-V cells were treated with 1 μM β-estradiol (Est) or 1 μM ATRA. Cell-cycle analysis was performed using propidium iodide staining at day 0 and 2. ▪ indicates the percentage of cells in G0-G1 stage, whereas □ indicates cells in G2-M stage and indicates cells in S phase. Median values are shown and error bars depict standard deviation from 3 independent experiments. (D) Wright-Giemsa staining of HT93A cells expressing the PU.1-ER fusion (HT-PER cells). The top 3 panels show unstimulated HT-PER cells (left panel), after 6 days of ATRA treatment indicating that the cells still differentiate toward neutrophils after having established the stable estrogen inducible system (middle panel), and after 6 days of treatment with 1 μM β-estradiol indicating differentiation toward neutrophils after 6 days (right panel). The bottom 3 panels depict the HT-V control line, which fails to show morphologic changes after β-estradiol treatment (right panel), but differentiates toward neutrophils after 6 days of ATRA treatment (middle panel). Images were visualized using a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany) and a 100 ×/2 numeric aperture objective. Images were acquired using a Zeiss AxioCam camera.

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