Increased IL-3 signaling in MLL-AF9 cells with mutated Jumonji domain and ZFD. (A) Western blotting of phospho- and total ERK1/2 and STAT5 in MLL-AF9-Cas9 leukemia cells 6 days after transduction with respective sgRNAs. Dose response curve (4-variable slope model) in the presence of RAS/ERK inhibitors (B) or JAK inhibitors (C). Two or 3 independent experiments, each done with technical triplicates. Shown are representative experiments (mean ± standard deviation [SD] of technical triplicates). Bar graphs on the far right show EC₅₀ (mean ± SD of 2-3 independent experiments) of inhibitors normalized to that of Renilla control. Numbers on top of the bar are average EC₅₀ (nM). *P < .05, **P < .01, unpaired 2-tailed Student t test. (D) Violin plot of relative gene expression data of Il3ra/Csf2rb/b2 derived from single-cell transcriptome. *****Adjusted P < .00001 by MAST. (E) Competitive cell-proliferation assay using sgRNAs against JMJD1C in the presence or absence of sgRNA against Csf2rb (marked by BFP). Data are mean ± SD of percentage of BFP⁺ and TdTomato⁺ double-positive cells from independently transduced triplicate samples. Curves were fitted with an asymmetric sigmoidal 5 parameter logistical model. (F) Model of transcription regulation of IL-3 receptor gene by JMJD1C.