KPT-SINE treatment causes decrease of CRM1 protein level, accumulation of CRM1 cargo proteins in the nucleus, and down-regulation of FLT3 and KIT oncoproteins. (A) Top panel: CRM1 protein expression as measured by Western blotting in MV4-11, Kasumi-1, and OCI-AML3 cells after KPT-185 treatment or control (DMSO) after 24 hours. Loading control is actin. Bottom panel: CRM1 protein expression as measured by Western blotting in 3 primary AML blasts after KPT-185 treatment or control. (B) Confocal microscopy of p53 in MV4-11 and OCI-AML3 cells treated with KPT-185 or control at 24 hours. Left panel: DAPI staining (cell nucleus). Middle panel: p53 staining. Right panel: merge of p53 and DAPI staining. Note the increase in the p53 expression in the nucleolus. (C) Whole-cell p53 protein expression in MV4-11 and OCI-AML3 cells (top panel) or primary AML blasts (n = 3) after KPT-185 or control treatment at 2 and 24 hours. (D) Confocal microscopy of NPM1 in OCI-AML3 cells and in a primary AML blast from a patient with CN-AML and NPM1 mutation treated with KPT-185 or control at 24 hours. Left panel: DAPI staining (cell nucleus). Middle panel: NPM1 staining. Right panel: merge of NPM1 and DAPI staining. The arrows indicate the localization of NPM1 in the cytoplasm (NPMc⁺) in the untreated samples and the elimination of the cytoplasmic signal on treatment with the drug, being detected exclusively in the nucleus. (E) FLT3 protein expression in MV4-11, Kasumi-1, and OCI-AML3 cells as measured by Western blotting after KPT-185 treatment or control at 24 hours. (F) FLT3 protein expression in primary AML blasts as measured by Western blotting after KPT-185 treatment or control at 24 hours: patient 1, CN-AML NPM1 WT, FLT3 ITD⁺; patient 2, CN-AML, NPM1 mutated, FLT3 WT; and patient 3, CN-AML, NPM1 mutated, FLT3 WT. (G) c-KIT expression in Kasumi-1 and OCI-AML3 cells after KPT-185 treatment or control at 24 hours.