SETBP1 combines with CSF3R mutations to promote cellular proliferation in vitro. (A) To evaluate the effects of SETBP1^WT, SETBP1^D868N, and CSF3R^T618I or the combination of these mutations on hematopoietic progenitors, mouse bone marrow was retrovirally transduced to express mutations of interest or appropriate retroviral control vectors. Cells were then sorted based on fluorescent markers and plated in cytokine-free methylcellulose media in triplicate for a CFU assay. Representative images are shown here at day 7. (B) Quantification of the colony phenotype shown in panel A. Statistics: 2-way analysis of variance (ANOVA) with Tukey correction, shown for key relationships. Both the CSF3R^T618I plus SETBP1^WT and CSF3R^T618I plus SETBP1^D868N groups were significantly higher than every group with an empty vector (P < .0001). (C) Individual colonies were harvested from the methylcellulose using a glass pipette and spread onto a glass slide. Slides were then allowed to dry for 4 to 6 hours, stained with May-Grünwald and Giemsa solutions, and imaged. Representative images shown for cells expressing CSF3R^T618I with either SETBP1^WT or SETBP1^D868N. (D) After 7 days in culture, cells were harvested by diluting the methylcellulose with PBS and performing 3 PBS washes. Cells were counted using a TC20, and ∼1250 cells per replicate per condition were plated into fresh cytokine-free methylcellulose media in triplicate. Serial replating was successful for at least 4 passages with both CSF3R^T618I plus SETBP1^WT and CSF3R^T618I plus SETBP1^D868N. (E) To evaluate if SETBP1^D868N enhanced colony formation driven by the endogenous ligand for CSF3R (granulocyte colony-stimulating factor [GCSF]), we plated 2000 SETBP1^D868N-expressing cells per well in cytokine-free methylcellulose media with or without exogenous GCSF (100 ng/mL). Representative images are shown. (F) Quantification of the CFU assay in panel E, with unpaired 2-tailed Student t test. (G) To determine if SETBP1^D868N enhances the CFU capacity of other CSF3R mutations, we transduced murine primary hematopoietic cells with either empty vector, CSF3R^WT, CSF3R⁷⁴¹*, CSF3R^T618I, or CSF3R^T618I/771fs (green fluorescent protein [GFP]) along with either empty vector or SETBP1^D868N (red fluorescent protein [RFP]). Representative images are shown. (H) Quantification of the CFU assay in panel G, with 2-way ANOVA for simple effects within group and Tukey correction for multiple comparisons. *P < .05, **P < .01, ***P < .001, ****P < .0001.