CSF3R^T618I- and SETBP1^D868N-expressing hematopoietic cells undergo cell-cycle arrest and differentiation after SETBP1 withdrawal. (A) Schematic of doxycycline (DOX)-inducible cell line generation (CSF3R^T618I plus SETBP1^D868N-dox). This cell line was generated from primary mouse bone marrow using retrovirally expressed oncogenes, with CSF3R^T618I in a constitutively active vector and SETBP1^D868N in a Tet-on vector. After transduction with both oncogenes, CSF3R⁺ cells (green fluorescent protein positive [GFP⁺]) were sorted and then cultured in the presence of DOX (1 μg/mL) to induce SETBP1^D868N expression. (B) Growth of CSF3R^T618I plus SETBP1^D868N-dox cells with and without DOX. To shut off SETBP1 expression, cells were washed with PBS 5 times and then resuspended in media with or without DOX in triplicate. Cells expressing only CSF3R^T618I stopped proliferating after 48 hours. (C) Cell death increased between 48 and 72 hours after SETBP1 withdrawal. (D) Representative flow cytometric plots for Cd11b and GR1 expression at 24 and 48 hours postwithdrawal. After withdrawing DOX, cells were collected at 24-hour intervals to monitor changes in cell state. (E) Quantification of Cd11b⁺GR1^high cells with and without DOX at 24, 48, and 72 hours. Statistics: repeated measures analysis of variance with multiple comparisons within time points [Šidák correction]. (F) Quantification of CD11b and GR1 at 48 hours, as gated in panel D. (G) Representative images of the cell line undergoing differentiation at 48 hours. ****P < .0001. PE, phycoerythrin.