Overexpression of SALL4-immortalized 32D cells and blocks G-CSF–dependent differentiation. (A) SALL4-induced expansion of 32D cells proliferate after the removal of IL-3 and addition of G-CSF. Three days after the removal of IL-3 and addition of G-CSF to the growth media of the cells, the SALL4A- and SALL4B-induced cells continue to expand, whereas the GFP-induced cells exhibit a decrease in cell number. At 7 days, the SALL4A- (i, 20×) and SALL4B-induced (ii, 20×) cells continue to proliferate, whereas the control cells (iii, 20×) have undergone cell death. (B) Growth curves of SALL4-induced 32D cells cultured only with G-CSF. 32D cells were transduced with SALL4A, SALL4B, or GFP lentivirus and then cultured for 3 days in growth media containing IL-3. On the day 4, 15 000 cells were aliquoted from each group and placed in new growth media with G-CSF and without IL-3. Cell growth was monitored daily, and the viable number of cells in each group was recorded. In cells that were transduced with SALL4A, an 8-fold increase in the number of cells was observed from day 1 to day 7. Cells that were transduced with SALL4B exhibited a 7-fold expansion of cells. In contrast, cells that were only transduced with GFP and wild type (WT, no lentiviral infection) demonstrated a decrease in the number of cells over the same period with almost all the cells undergoing cell death by day 5. (C) Wright-Giemsa staining of 32D cells (100×). Morphology of 32D cells with IL-3 alone (iv), transduced with SALL4A with G-CSF (v), and with G-CSF alone (vi). Cells given IL-3 or transduced with SALL4A continue to demonstrate blastlike morphology (iv and v), whereas the cells not transduced with SALL4A and given G-CSF exhibit neutrophil morphology (vi).