RA and arsenic induce proteasomal degradation of mutant NPM1 and restore NPM1 nucleolar localization. (A) Western blot analysis for NPM1 recognizing both WT and mutated NPM1 (WT + c), mutated NPM1 (NPM1c), actin in THP-1, and OCI-AML3 cells treated with arsenic (1 μM), RA (1 μM), or a combination of both for 48 hours as indicated. A representative of 3 independent experiments is shown. (B) Western blot analysis for NPM1 (WT + c) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in primary leukemic cells derived from AML patients treated with arsenic (0.1 or 1 μM), RA (0.3 or 1 μM), or a combination of both for 48 hours as indicated. (C) Western blot analysis for NPM1 (WT + c) and GAPDH in OCI-AML3 cells treated with arsenic (1 μM), RA (1 μM), PS-341 (10 nM), either alone, or in combination for 48 hours as indicated. Percentages indicate the amount of remaining NPM1 (WT + c) after normalization to GAPDH. (D) Cell count with trypan blue staining (percent of control) of OCI-AML3 and THP-1 cells treated with arsenic (1 μM), RA (1 μM), PS-341 (10 nM), either alone, or in combination for up to 48 hours. Cell growth (percent of control) was assayed in triplicate wells. The results depict one representative experiment among 3 independent ones. (E) Confocal microscopy analysis of nucleolar NPM1 localization in OCI-AML3 or THP-1 cells after treatment with RA/arsenic for 48 hours. NPM1 was stained with an antibody recognizing NPM1 (WT + c) (green), nucleoli were stained with anti-Fibrillarin (red), and nuclei were stained with 4,6 diamidino-2-phenylindole (blue). Images represent Z sections. Graphs show quantification of nucleolar NPM1 as averages of one Z section/cell from 30 different cells of 3 independent experiments. Significant P values are indicated by asterisks.