ATO/ATRA induce proteasome-dependent NPM1 mutant oncoprotein downregulation. (A) Western blot analysis of NPMm and NPMwt protein levels upon ATO (left) and ATRA (right) treatment in OCI/AML3. Active caspase-8 and cleaved PARP (cPARP) indicate apoptosis activation (right). (B) Western blot analysis of NPM1 mutant and wild-type protein levels upon ATO, ATRA, and ATO/ATRA combination at lower (left) and higher doses (right) in OCI/AML3. (C) Analysis by western blot of NPM1 mutant proteins levels upon ATO, ATRA, or ATO/ATRA combination on OCI/AML3 cells pretreated with proteasome inhibitor MG132. Band intensity quantification (NPM1 mutant/H3 ratio) from 3 independent experiments (mean ± SD). (A-C) Results are representative of at least 3 independent experiments. (D-E) Western blot analysis of the effects of ATO on NPM1 mutant protein levels in 11 primary NPM1-mutated AML patient cells. Downregulation of NPM1 mutant oncoprotein upon ATRA treatment in 4 NPM1-mutated AML patients is shown in panel E. Phosphorylation of histone H2AX (γH2AX) is expression of DNA damage. (A-E) Antibodies recognizing specifically the NPMm protein or NPMwt protein have been used, as described in “Methods.” Histone H3, β-tubulin, or β-actin expression was used as loading control. In all cases, membranes probed with the different antibodies were from the same gel. Vertical lines have been inserted in blot images to indicate repositioned lanes within the same gel.