Cell experiment:

HEK 293 cells stably expressing NFAT-GFP are grown on glass coverslips to approximately 50% confluency prior to drug treatment. In all cases, importazole is used at 40 μM and leptomycin B is used at 10 ng/mL. For controls, DMSO is used at a concentration of 0.4%. Ionomycin is added at 1.25 μM. Importazole and leptomycin B treatments are all for 1 hour. In all experiments cells are fixed with 4% formaldehyde prior to fluorescence microscopy. DNA is visualized with 1 μg/mL Hoechst dye. For quantification, 100 cells from each condition are analyzed and the percentage that shows nuclear accumulation of NFAT-GFP are calculated[1].

Importazole is a small molecule inhibitor of the nuclear transport receptor importin-β.

Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. Importazole impairs the release of an importin-β cargo FRET probe and causes both predicted and novel defects in spindle assembly[1]. Importazole displays an IC50 of approximately 15 uM for inhibition of NFAT-GFP import. Importazole has an IC50 of approximately 22.5 uM in HeLa cells following treatment over a 24-hour period[1]. Importazole induces a dose-and time-dependent inhibition of myeloma cells growth. And the IC50 values of importazole on RPMI 8226 and NCI-H929 after 48 hours incubation are (4.43±0.41) and (4.78±0.35) umol/L, respectively. Treatment of RPMI 8226 and NCI-H929 cells with 8 umol/L importazole for 24 h could inhibit NF-κB import to nucleus and reduce its DNA binding activity[2].

References:
[1]. Soderholm JF, et al. Importazole, a small molecule inhibitor of the transport receptor importin-β. ACS Chem Biol. 2011 Jul 15;6(7):700-8.
[2]. Yan WQ, et al. Effect of nuclear receptor inhibitor importazole on the proliferation and apoptosis of multiple myeloma cells. Zhonghua Xue Ye Xue Za Zhi. 2013 Apr;34(4):323-6.