Induced expression of Dusp1 in CSF3R mutant expressing cells. (A) Heat map showing deregulated expression of MAPK pathway genes in BM-derived Kit⁺ cells expressing different CSF3R mutants. Total RNA from the venus-positive Kit cells after 24 hours of transduction were subjected to RNA-seq analysis.⁹ Total RNA from the vector (pMSCV-Ires-venus) transduced Kit⁺ cells was used to filter out differentially expressing cells. Differences in the levels of expression were measured using t test between the samples from the leukemic variants (CSF3R^T618I and CSF3R^{T618I/Q741∗)} and nonleukemic truncation mutants (CSF3R^Q741∗ and CSF3R^W791∗). (B) A bar graph showing the relative expression of Dusp1 in Lineage-negative, KIT⁺, and SCA1⁺ cells expressing CSF3R mutants. (C) Immunoblots from the total protein extracts of control (sc-shRNA) and Dusp1-depleted BaF3 cells expressing CSF3R mutants grown without IL-3 showing reduced p-ERK1/2 and induced activation of p-JNK1/2 upon Dusp1 knockdown in cell expressing leukemic CSF3R mutants (CSF3R^T618I, CSF3R^{T618I/Q741∗}, and CSF3R^T618I/W791). In contrast, the nonleukemic CSF3R truncation mutations exhibit modest elevation in p-ERK1/2 without any alteration in p-JNK1/2 levels. Expression levels were quantified and normalized to the control condition (pMIV-Vector expressing Sc-ShRNA normalized to to β-actin). The resulting normalized values are presented below each blot for reference. BaF3 cells expressing Dusp1-shRNA resulted in ∼6% to 70% knockdown at protein level and control sc-shRNA has been described earlier.¹⁴ (D) A cell proliferation growth curve of Dusp1-depleted BaF3 cells expressing CSF3R mutants showing normal growth when grown with IL-3. (E) Growth curve showing significantly reduced proliferation upon Dusp1 depletion in the absence of IL3. This assay revealed that the transformation potential of CSF3R is compromised upon Dusp1 depletion. Presented data are from 2 independent experiments, shown as means ± standard deviation (SD). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. RNA-seq, RNA sequencing.