were treated with 5 mM Cell Tracker Green (Invitrogen, Carlsbad, CA) in serum-free media in suspension at 37uC. After 1 hr Panc-1 cells were collected by centrifugation, washed in PBS and then mixed with unlabeled Panc-1/C5 cells (16106) and plated onto glass coverslips in complete media. After 24 hr the coverslips were fixed for immunocytochemisty as described below. Achieved formalin fixed human squamous lung cancer tissue was obtained from the University of Colorado Cancer Center and immunostained for NQO1 as previously described [18]. Immunocytochemistry Immunocytochemistry was performed on cell lines grown on glass coverslips using the following methods. All steps were performed at room temperature. Cells were washed in phosphate buffered saline (PBS) then fixed in 3.7% (v/v) formaldehyde in PBS for 12 min, rinsed with PBS, then permeabilize with 0.15% (v/v) Triton-X100 in PBS for 10 min. Cells were then blocked in RPMI1640 containing 5% fetal bovine serum for 1 hr at room temperature. Coverslips were then transferred to a 6-well plate (single coverslip/well) and primary antibodies diluted in TBST (10 mM Tris-HCl, pH 8 containing 150 mM NaCl and 0.4% (v/ v) Tween-20), were added (2 ml/well). After 90 min the primary antibodies were removed, the cells were gently rinsed with TBST, and secondary antibodies (diluted in TBST) were then added (2 ml/well) for 30 min. DAPI (1 mg/ml) was included with the secondary antibody. After 30 min coverslips were rinsed in TBST, briefly immersed in deionized/distilled H20, inverted, and mounted on to glass microscope slides using SuperMount (Biogenex, San Ramon, CA). Stained cells were examined using References 1. Lind C, Cadenas E, Hochstein P, Ernster L (1990) DT-diaphorase: purification, properties, and function. Methods Enzymol 186: 287–301. 2. 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When unconditioned RPMI1640 containing 10% fetal bovine serum was substituted for conditioned hybridoma clone A180 supernatant no immunostaining of cells or mitotic spindles could be observed. Panel A, acquired with confocal settings used for Figure 1; Panel B, acquired with confocal settings greatly enhanced for detection of Dylight 549 nm (red). Arrows indicate mitotic cells. (TIF) Figure S2 NQO1 immunostaining in BxPc-3 cells using hybridoma supernatant or purified antibody. Identical immunostaining of NQO1 in BxPc-3 cells was observed using either (A) hybridoma tissue culture media from clone A180 diluted 1:10 or (B) affinity purified mouse IgG from clone A180 (Cell Signaling Technologies) diluted 1:200. (TIF) Author Contributions Conceived and designed the experiments: DS JK DR. Performed the experiments: DS. Analyzed the data: DS JK DR. Contributed reagents/ materials/analysis tools: JK. Wrote the paper: DS JK DR. 14. 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