p53 against 20 S proteasomal degradation [16]. In experiments designed to monitor the subcellular distribution of NQO1 in human cells by confocal microscopy we discovered that NQO1 could also be found in association with the mitotic spindle. Results Immunofluorescent staining of the human pancreatic adeno- carcinoma cell line BxPc-3 for NQO1 revealed that NQO1 is located primarily in the cytosol of these cells (Fig. 1). However, in BxPc-3 cells undergoing mitosis intense immunostaining for NQO1 was observed on the mitotic spindles (Fig. 1). Our source of anti-NQO1 monoclonal antibody for these studies was conditioned tissue culture supernatant from mouse hybridoma clone A180. In control studies, we utilized unconditioned media (RPMI1640 containing 10% fetal bovine serum) in place of hybridoma clone A180 supernatant and in these studies no immunofluorescent staining of NQO1in BxPc3 cells or spindles could be observed (Fig. S1). In addition, no observable difference in immunostaining of NQO1 including spindles could be detected in BxPc-3 cells between tissue culture supernatant from hybridoma clone A180 or commercially prepared affinity purified mouse IgG from hybridoma clone A180 (Fig S2). To confirm that NQO1 was the target of monoclonal antibody A180 we generated a BxPc-3 cell line stably expressing a doxycycline-inducible anti-NQO1shRNA expression vector. The growth of this genetically modified BxPc3 cell line in doxycyline for 7 days resulted in near complete knockdown of NQO1 protein expression as confirmed by immunoblotting for NQO1 (Fig 2). As expected, doxycycline treated cells showed no detectable immu- nostaining for NQO1 either in the cytosol or on mitotic spindles (Fig 2). These data confirm that NQO1 is the target of monoclonal antibody clone A180 and that the immunostaining of mitotic spindles by anti-NQO1 antibodies from clone A180 is not observed when NQO1 protein levels are knockdown. To confirm co-localization of NQO1 with the microtubule containing mitotic spindles, doubling-labeling experiments were performed using antibodies against NQO1 and alpha-tubulin and results from these studies confirmed the co-localization of NQO1 with alpha-tubulin in the mitoic spindles (Fig 3). The association of NQO1 with the mitotic spindles could be observed throughout mitosis from metaphase to cytokinesis (Fig. 3). In cells not undergoing division no obvious co-localization of NQO1 with alpha-tubulin was observed, (see Fig. 3 D–F) suggesting that the association between NQO1 and microtubules is specific for mitotic NQO1 Localizes to the Mitotic Spindle spindles. Treatment of BxPc-3 cells with the competitive inhibitor dicoumarol or the mechanism-based inhibitor ES936 did not prevent the association of NQO1 with mitotic spindles suggesting that the association of NQO1 with the mitotic spindle is not dependent upon the redox state of NQO1 (Fig. 4). The immunofluorescent staining of NQO1 was performed in many different human cell lines (Fig. 5) including cancer cell lines (pancreatic), immortalized cell lines (HBMEC, 16HBE) and primary cell lines (astrocytes, HUVEC). The human pancreatic cancer cell line Panc-1, which has previously been genotyped as homozygous for the NQO1*2 polymorphism [17] and character- ized as NQO1 null, did not demonstrate detectable immunoflu- orescent staining for NQO1 either in the cytosol or mitotic spindles. Stable expression of NQO1 in the Panc-1 cell line (clone panc-1/C5) resulted in significant staining for NQO1 in the cytosol and mitotic spindles (Fig 6). To determine if NQO1 could be found associated with mitotic spindles in human tissues, we examined human NSCLC tissue samples that had been immunostained for NQO1 in a previous study [18]. Upon examination of squamous cell carcinomas samples mitotic figures were located and immunostaining for NQO1 was clearly visible on the mitotic spindles of these cells (Fig 7). These data support our studies in cell culture that demonstrate the association of NQO1 with mitotic spindles. Discussion These data demonstrate that the quinone oxidoreductase NQO1 associates with the mitotic spindles and suggests that this association with the mitotic spindle is independent of the redox state of NQO1 since immunostaining for NQO1 on spindles was not diminished in the presence of NQO1 inhibitors. While this is the first report of localization of NQO1 with mitotic spindles, previous studies using Xenopus egg extracts indentified NQO1 as a target in a chemical screen of purine-based mitotic disrupters [19]. In this study, diminutol (2-(1R-isopropyl-2-hydroxyethyla- mino)-6-(3-aminophenylthio)-9-isopropylpurine), which prevents microtubule polymerization, was also shown to inhibit NQO1 catalytic activity via competitive binding with NAD(P)H. These same studies demonstrated immuno-depletion of NQO1 from Xenopus egg extracts resulted in defects in spindle assembly [19]. Despite these findings that suggested NQO1 catalytic activity may be linked to spindle assembly in Xenopus eggs, the authors did not demonstrate localization of NQO1 with spindles [19]. Unlike studies in Xenopus eggs, our studies in human cells have shown Figure 1. Immunofluorescent staining of NQO1 in BxPc-3 human pancreatic cancer cells. Immunostaining for NQO1 reveals cytosolic localization and intense staining of mitotic spindles in cells undergoing division (*). doi:10.1371/journal.pone.0044861.g001 PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44861