Fig. 7.

Downregulation of ENT1 by KDM6A loss is regulated by H3K27 acetylation and increases AraC resistance. a Volcano plot showing log₂ fold change on the x-axis and adjusted P value on the y-axis for the differential gene expression between shRNA-mediated KD of KDM6A (shKDM6A #7) and control (shGFP) in K562 cells (n = 6). Genes with adjusted P value < 0.05 are highlighted in red and those with a log₂ FC >2.5 or <−2.5 are labeled with the gene name. In addition, genes with adjusted P value < 1e-8 and the gene SLC29A1 are labeled. b qRT-PCR for ENT1 in K562 cells: native, shRenilla, shGFP and three different shKDM6A KDs. ENT1 mRNA for shRenilla, shKDM6A #3 and shKDM6A #7 K562 cells is also shown after treatment with 150 nM AraC for 72 h. The mean ± s.d. for ENT1 mRNA relative to GAPDH for three independent experiments is shown. c Immunoblotting showing strong reduction of ENT1 protein in KDM6A mutant MM-6 cells compared to the KDM6A WT sister cell line MM-1. Immunoblots are representative of three independent experiments. MW, molecular weight; β-actin, loading control. d Inhibition of ENT1 by NBMPR increases the amount of viable cells during AraC treatment. K562 cells were treated with different AraC concentrations in combination with 0, 0.1, 1 and 10 μM of NBMPR for 72 h. Mean ± s.d are given for three independent experiments. Unpaired, two-tailed Student’s t-test; *P < 0.05; **P < 0.01; ***P < 0.001. e Genomic snapshot of H3K27ac ChIP-seq in K562 KDM6A WT, K562 KDM6A KO PB KDM6A and KDM6A mutant THP-1 cells in the absence or presence of doxycycline at the ENT1 locus. Cells were treated with media +/− doxycycline (0.5 μg/mL) every 24 h for 72 h