Figure 9. Proposed mechanisms for TSA-induced apoptotic cell death in NQO1-positive cells. TSA induces apoptotic cell death of NSCLC cells via a unique NQO1-initiated and ROS-mediated activation of a p53 independent but caspase dependent mitochondrial apoptotic pathway. doi:10.1371/journal.pone.0042138.g009 Synergy-H1 fluorimeter (Bio-Tek Instruments); the changes in mitochondrial potential were calculated as the red/green ratio for each condition. Caspase activity analysis Caspase-3, caspase-8, and caspase-9 activities were measured by colorimetric assays kits (KeyGen, Nanjing, China). Cells post-treatment were lysed in chilled lysis buffer and were centrifuged at 15,000 g for 5 min; the supernatants were added to 50 mL of reaction buffer and 5 mL of respective caspase substrate. After incubation at 37uC for 4 h, absorbance was read at 405 nm. Transient NQO1 or P53 siRNA transfection A549 cells were transfected with control siRNA or siRNA targeting NQO1 or P53 (invitrogen) using RNAiMAX transfec- tion Reagent (Invitrogen) according to the manufacturer’s instruction. Two days later, cells were harvested for western blot analysis to confirm the efficiency of siRNA knockdown. Western blot analysis Cells were gently scraped with cell scrapers and subjected to total protein extraction. Protein concentration was then deter- mined using the Bradford method. Equal amount of protein (50 mg) was loaded and separated by SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane (PALL, USA). Blots were blocked with 5% skim milk in TBST buffer and incubated overnight at 4uC with specific primary antibodies. After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody (KeyGen, Nanjing, China) for 1 h. The signal was visualized by enhanced chemiluminescence (ECL, Millipore). The protein expression levels were normalized with GAPDH or COX-IV to correct any experimental handing error. TSA as an NQO1 Specific Target Agent Subcellular fractionation Cells were harvested and washed in ice-cold PBS and then resuspended in an isotonic buffer on ice for 20 min. Cells were then homogenized and centrifuged at 8006 g for 10 min at 4uC. Supernatants were centrifuged at 14,0006 g for 30 min at 4uC to obtain mitochondrial and cytosolic fractions. Mitochondrial fractions were lysed in 1% Chaps buffer for immunoblot analysis. In vivo anti-tumor efficacy Log-phase A549 cells (56106) were injected s.c. into the flanks of athymic nude mice (aged 6–8 weeks). Tumor sizes were regularly measured using calipers, and volumes were calculated using the following formula: volume (mm3) = length6width6width/2. Ani- mals were randomized into four groups (6 mice/group) when the average tumor size reached around 200 mm3 for the following treatments: group 1, vehicle; group 2, DIC (5 mg/kg); group 3, TSA (20 mg/kg); and group 4, DIC (5 mg/kg) 1 h prior to TSA (20 mg/ kg) injection. All agents were administered every other day for 3 weeks through intraperitoneal administration. Tumor sizes were monitored and measured every other day. Differences in tumor volumes were analyzed statistically by comparing the slopes of the regression lines for plots of tumor volume vs. days of TSA treatment in mice receiving different treatments. Biodistribution study in A549 tumor inoculated nude mice Mice randomized to six groups were sacrificed at 2 or 8 h after a single intraperitoneal administration of TSA at 20 mg/kg or a co-administration of 5 mg/kg of DIC with TSA (20 mg/kg), respectively. TSA levels in the plasma, major organ tissues, and tumors were determined by a LC-MS/MS method as previously reported [53]. Statistical analysis All data are presented as means 6 SE of at least three samples and reproducibility was confirmed in three independent experi- ments. Statistical differences between two groups were evaluated using the Student’s t-test; for multiple comparisons, one way analysis of variance followed by Student-Newman-Keuls Post Hoc test was applied. The difference was considered significant at *P,0.05, **P,0.01, or ***P,0.001. Supporting Information Figure S1 TSA induced time dependent increased ratio of Bax to Bcl-xl. A549 cells were treated with 10 mM of TSA for indicated time, whole cell lysates were prepared and western blot were conducted using anti-Bax, anti-Bcl-xl and anti-GAPDH antibodies. The relative density value of each band is shown below the Western blot. The data are representative of a typical experiment that was conducted three times (mean values, P,0.05). (TIF) Figure S2 Time and concentration dependent intracel- lular accumulations of TSA in NSCLC cells. A, H596 versus A549 cell lines; B, control siRNA versus NQO1 siRNA transfected A549 cells. Confluent monolayer cells were preincu- bated in 1 mL of HBSS at 37uC for 30 min and then washed twice with HBSS before cellular uptake assay. Time dependent experiments were tested at TSA concentration of 10 mM and incubated for 15–210 min; concentration dependent accumula- tions were determined at TSA concentration range of 0–40 mM for 120 min. At the indicated time-points, cells were collected and lysed for determining the intracellular concentration of TSA by a PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e42138