RNA-seq analysis identifies increased expression of OXPHOS and ROS gene signatures in TKI nonresponder cells and PAK6 as a target of miR-185. (A) DESeq2 analysis of differentially expressed genes from the same 9 RNA samples used for miRNA profiling and a comparison of genes expressed differentially in CD34⁺ cells from NBM (n = 3) vs CML (n = 6, top panel) samples and IM responder (n = 3) vs IM nonresponder (n = 3, bottom panel) samples. Red dots represent differentially expressed genes with adjusted P values < .05 between these samples. (B) Gene set enrichment analysis plots for OXPHOS and ROS pathways, with nominal enrichment scores. (C) Heat maps of miR-185-predicted target genes identified in CD34⁺ cells from NBM vs CML cells. (D) qRT-PCR analysis of miR-185 expression in IM-resistant K562R cells, CD34⁺ IM responder (n = 3) and IM nonresponder (n = 3) cells transduced with a miR-185-expressing vector or a pRRL control vector and incubated with or without IM (5.0 µM) for 24 hours. (E) Western blot analysis of several key signaling proteins in miR-185-transduced K562R cells, PAK6 siRNA-transfected K562R cells, and control cells cultured with or without IM (5.0 µM) for 24 hours. ACTIN served as loading control. (F-H) qRT-PCR analyses of the transcript levels of PAK6 in CD34⁺ cells from NBM vs IM responders (R) or IM nonresponders (NR). Correlation analysis of PAK6 and miR-185 expression in responder and nonresponder patients (G), and PAK6 transcript levels in CD34 subpopulations from responders vs nonresponders. P values were calculated using a 2-tailed paired (Figure 6F) or unpaired Student t test. Ctrl = control.