NQO1 Localizes to the Mitotic Spindle Figure 6. Co-localization of NQO1 with mitotic spindles is not observed in cells homozygous for the NQO1*2 polymorphism. Panc-1 cells which are homozygous for the NQO1*2 polymorphism (NQO1 null), were prelabeled with cell tracker green then mixed with unlabled Panc-1/C5 cells which stably express wild type hNQO1. Following a 24hr coincubation period cells were fixed and immunostained for NQO1 followed by confocal microscopy. Cells were searched until a mitotic Panc-1 cell (green) and a mitotic Panc-1/C5 cell (non-staining) were observed in the same field. Panels A and C, data was collected using 488nm excitation wavelength for cell tracker green fluorescence (green Panc-1 cells). Panels B and D, data was collected using 561nm excitation wavelength (red/NQO1). Co-localization of NQO1 to the mitotic spindles was not observed in NQO1-null Panc-1 cells (green) while co-localization of NQO1 to the mitotic spindle was seen in NQO1 overexpressing Panc-1/C5 cells. doi:10.1371/journal.pone.0044861.g006 streptomycin. All cells were maintained as monolayers at 37uC in 5% CO2. For immunocytochemical studies all cells were plated onto glass coverslips (18 mm60.15 mm circle, Fisher Scientific) in 100 mm tissue cultures dishes 3 days prior to analysis. For studies using NQO1 inhibitors ES936 and dicoumarol cells were incubated in fresh media (2 ml) containing inhibitors (500 nM ES936 or 50 mM dicoumarol) for 2 hr. The generation of a doxycycline-inducible NQO1 knock-down cell line was performed using a TRIPZ inducible shRNA plasmid for human NQO1 (Open Biosystems, Lafayette, CO). To create a stable knockdown cell line, 26106 BxPc3 cells were electroporated with 20 ug of TRIPZ inducible shRNA plasmid for human NQO1 and transfected cells were selected in normal culture media containing 3 mg/ml puromycin. The antibiotic resistant cells were induced with 300 ng/ml doxycyclin for 48 h and then cells were sorted for turbo red fluorescence (553excitation/574emission) using the Legacy MoFlo sorter (University of Colorado Cancer Center). Sorted cells were maintained in complete media containing 500 ng/ml doxycycline and knockdown of NQO1 was confirmed by immunoblot analysis. Coincubation studies with Panc-1 cells and Panc-1/C5 cells were performed using the following method. Panc-1 cells (16106) Figure 7. NQO1 localizes to mitotic spindles in human lung cancer cells. Immunohistochemical analysis of NQO1 (DAB, brown) in archival formalin-fixed human squamous lung carcinoma tissue samples. Mitotic figures are visible due to their intense chromatin staining (hemotoxylin). doi:10.1371/journal.pone.0044861.g007 PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44861