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 (*).
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.
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.
Figure 4.
NQO1 inhibitors do not prevent binding of NQO1 to mitotic spindles.
Immunostaining for NQO1 (red) in BXPC3 cells pretreated with DMSO (panel A), ES936 (500 nM, panel B) or dicoumarol (50µM, panel C). Cells were pretreated with inhibitors 2 hr before immunostaining. Arrows indicate mitotic cells.
Figure 5.
Co-localization of NQO1 and alpha-tubulin with mitotic spindles in human cells.
Triple immunofluorescent staining for NQO1 (red, panel A); alpha-tubulin (green, panel B); merged (with DAPI,panel C).
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.
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).