NQO1 Localizes to the Mitotic Spindle Figure 2. Knockdown of NQO1 eliminates immunostaining of NQO1 in BxPc-3 cells. Knockdown of NQO1 in BxPc-3 cells was performed using a doxycyline- inducible anti-NQO1 shRNA methodologies. BxPc-3 cells were immunostained for NQO1 (green) in the absence (panel A) and presence (panel B) of doxycyline pretreatment. DAPI staining was included in panel B to aid in the location of cells. Right panel: immunoblot demonstrating near complete knockdown of NQO1 protein in the presence of doxycycline. Arrows indicate mitotic cells. doi:10.1371/journal.pone.0044861.g002 that NQO1 can be found associated with the mitotic spindles; however, treatment of human cells with the NQO1 mechanism- based inhibitor ES936, which resulted in .95% inhibition of NQO1 catalytic activity, did not alter the growth rate of these cells [20]. These data suggest that in human cells despite the binding of NQO1 to the mitotic spindles NQO1 most likely does not play a direct role in regulating mitosis. These observations are reinforced by studies in human cell lines that lack NQO1 activity and protein due to homozygous expression of the NQO1*2 polymorphism. Many commonly used human cells lines (BE, MDA231, MDA468, Panc-1, Caco-2) lack NQO1 activity and protein due to homozygous expression of the NQO1*2 polymorphism and for many of these cell lines isogenic pairs have been created following the stable transfection of wild type NQO1. The reintroduction of wild type NQO1 back into these cell lines did not significantly alter the growth rate when compared to their NQO1 isogenic parental cells [21]. Taken together, these data imply that in human cells NQO1 does not play a major role in regulating mitosis but instead NQO1 may act in a protective role. Figure 3. Co-localization of NQO1 and alpha-tubulin in mitotic spindles and midbody region in BxPc-3 human pancreatic cancer cells. Triple immunofluorescent staining for NQO1 (red), alpha-tubulin (green) and nuclei (blue). Association of NQO1 with spindles can be seen in different stages of mitosis including metaphase (*), telophase (**) and the midbody region (arrow) during cytokinesis. doi:10.1371/journal.pone.0044861.g003 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44861