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. 2016 Dec 1;7(12):e2497. doi: 10.1038/cddis.2016.392

Figure 4.

Figure 4

Avrainvillamide treatment causes nuclear retention of NPMc+ proteins and reduced expression levels of NPMc+, CRM1 and FLT3 proteins in AML cell lines. (a) OCI-AML3 cells were treated DMSO (CTR) or avrainvillamide (AVA; 0.5 μM) for 24 h or LMB (10 nM) for 3 h, then cytospun onto coverslips, fixed, and stained with NPM1wt- and NPMc+-specific antibodies. (b) MV4-11 cells were treated with DMSO (CTR) or AVA (0.5 μM and 1.0 μM), cytospun onto coverslips, fixed, and stained with NPM1wt and RanBP1 antibodies. All immunofluorescence experiments were conducted at least three times; representative images are shown and arrows indicate described localization and zoomed cells for MV4-11. (DAPI indicates nuclei; scale bar, 10 μm). (c) OCI-AML3 and MV4-11 cells were treated with AVA (0.5 μM) for 24 h and analyzed by flow cytometry using specific antibodies against NPMc+ and NPM1wt as indicated. Results from representative experiments are shown. (Sec=secondary antibody only). (d) Flow cytometry median fluorescence intensity (MFI) quantification of NPM1 expression levels in OCI-AML3 and MV4-11 cells. Results represent the median±S.E.M. of three independent experiments (**P<0.01). (e) Validation of NPM1 antibody specificity by immunoblotting using cell lysates of OCI-AML3 (OCI) and MV4-11 (MV4) incubated with NPM1wt and NPMc+ antibodies. (f) Immunoblots for NPMc+ expression in OCI-AML3 cells and NPM1wt expression in OCI-AML3 and MV4-11 cells incubated with AVA (0.5 μM) for 24 h. (g) CRM1 expression as determined by flow cytometry in OCI-AML3 and MV4-11 cells after incubation with AVA (0.5 μM) for 24 h, representative image is shown and (h) flow cytometry MFI quantification of the median±S.E.M. of three independent experiments relative to control (n=3, **P<0.01; Sec=secondary antibody only). (i) CRM1 expression after incubation with AVA (0.5 μM; OCI-AML3, 1.0 μM; MV4-11) for 24 h, as determined by immunoblotting. (j) FLT3 expression after incubation with AVA (0.5 μM; OCI-AML3, 1.0 μM; MV4-11) for 24 h, as determined by immunoblotting. (k and l) OCI-AML3 cells were treated with either DMSO (CTR), AVA alone (0.5 μM, 24 h), AVA (0.5 μM, 24 h) with bortezomib (Comb; 50 nM, final 8 h) or bortezomib (Brz; 50 nM, final 8 h) alone and then immunoblotted for NPMc+, CRM1, FLT3 and NPM1wt. Actin and CoxIV were used as loading controls for the immunoblots