TSA as an NQO1 Specific Target Agent Figure 8. TSA retards the tumor growth NQO1 dependently in A549 tumor xenografted nude mice. A, upper, tumor volume measurement (P,0.01, TSA vs. control; P,0.05, TSA vs. TSA+DIC); lower, tumor weight (# P,0.05, TSA vs. TSA+DIC, *** P,0.001, TSA vs. control); data are shown as mean 6 SE of six mice. B, histological examinations; tumor and tissue sections were analyzed by hematoxylin and eosin staining for histological features. C, biodistribution of TSA in A549 tumor loaded nude mice; data are shown as mean 6 SE of 6 mice. doi:10.1371/journal.pone.0042138.g008 solution was removed and 150 mL DMSO was added per well; the absorbance at 570 nm was measured. Apoptosis assay Apoptosis was quantified by using APO-BRDUTM Kit (BD Pharmingen, USA) according to the manufacturer’s instruction. Samples were analyzed in a flow cytometer (BD FACSCalibur, USA) using an air-cooled argon laser at 488 nm. In view of the potential EGFP interference in NQO1 transfected H596 cells, the parallel TUNEL assay of H596-vector and H596-NQO1 cells were conducted using Click-iT TUNEL Alexa Fluor 594 Imaging Assay Kit (Molecular Probes, USA). Imaging was performed with Leica BMI3000 B microscope with a confocal microscope application. ROS assay Intracellular ROS was analyzed from the conversion of nonfluorescent DCFH-DA to its fluorescent derivative. Fluores- cence intensity was detected at 535 nm (with 488 nm excitation) in Synergy-H1 fluorimeter (Bio-Tek Instruments). Total glutathione and GSSG were measured from the reduction rate of 5,59-dithio- bis (2-nitrobenzoic acid) (DTNB) using a commercial assay kits (Beyotime, Jiangsu, China). The absorbance changes at 412 nm were detected in an ultraviolet/visible spectrophotometer. GSSG to GSH ratio was calculated as an indicator of the intracellular oxidative stress. Alkaline Comet Assays DNA lesions in single cells, including DNA single- and double- strand breaks, were assessed by alkaline comet assays. Slides were stained with ethidium bromide and visualized by using a fluorescence microscope (Nikon DS 5 M U1 CCD, Japan). Digital photomicrographs were taken and comet tails were quantified by analyzing at least 50 randomly selected comets per slide with the software (Resarch Biolab Co., ltd, Beijing, China). Mitochondrial Transmenbrane Potential measurement The changes in mitochondrial membrane potential were monitored by 5,59,6,69-tetrachloro-1,19,3,39-tetraethyl-benzimida- zolcarbocyanine iodide (JC-1) (KeyGen, Nanjing, China) staining. The green monomeric form of this dye in the cytosol and the red concentrated aggregates in the mitochondria were detected using a PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e42138