Cytoplasmic RAN contributes to BCR-ABL1 kinase-independent TKI resistance. (A) Whole cell, nuclear, and cytoplasmic lysates of K562^S, K562^R, AR230^S, and AR230^R cells (n = 3) either untreated or treated with 1 μM imatinib were separated by SDS-PAGE and analyzed for RAN expression and subcellular localization by immunoblot analyses. (B) qRT-PCR quantitation of RAN mRNA in shRAN-expressing cells (n = 3) either untreated or treated with doxycycline (72 hours, 0.1 μg/mL). (C) shRAN-induced apoptosis was assessed by staining with annexin V followed by flow cytometric analysis (n = 6). (D) Parental and TKI-resistant K562 and AR230 cells were incubated with and without doxycycline at graded imatinib concentrations, followed by quantification of viable cells by MTS assay at 72 hours (n = 3). (E) Table shows imatinib IC₅₀ (nM) of K562^R and AR230^R cells expressing shRAN in the presence or absence of 0.1 μg/mL doxycycline, as measured by MTS assay following treatment of 72 hours. Error bars represent SEM; *P < .05.