Signaling. Rabbit polyclonal anti-caspase 3 (1:1 000) was from Stressgen (Victoria, Canada) and mouse monoclonal anti-c-FLIP (NF6, 1:500) was from Alexis Biochemicals. Rabbit polyclonal anti- DR4 (1:1 000), anti-DR5 (1:1 000) and anti-TRAIL (1:500) were purchased from Chemicon International Inc. Protein blots were probed with mouse monoclonal anti-b-actin (AC-15, Sigma) as controls for protein loading. Bound primary antibodies were detected using HRP-conjugated secondary antibodies: anti-rabbit IgG (1:10 000) or anti-mouse IgG (1:10 000) provided from BD Pharmingen. Proteins were visualized by using enhanced chemilu- minescence detection method (GE Healthcare) followed by film exposure (Hyperfilm ECL, GE Healthcare). Assessment of cell surface DR4, DR5 and TRAIL expression Cells were treated with ciglitazone alone or in combination with TRAIL as described above. They were detached with 5 mM EDTA and centrifuged at 1 200 rpm for 10 min. Cells (1.106) were resuspended in 50 mL of PBS and stained with 10 mL of phycoerythrin (PE)-conjugated mouse monoclonal anti-human DR4 (clone B-N36), DR5 (clone B-K29) or TRAIL (clone B-S23) provided by Diaclone (Besanc¸on, France), for 30 min at 4uC in the dark. Cells were then washed twice in 1% BSA-PBS and resuspended in 50 mL of PBS. Flow cytometry analyses were performed at a 580 nm wavelength. PE-conjugated mouse IgG1 was used as an isotype control. Determination of TRAIL production RT4 and T24 cells were seeded in triplicates in 6-well plates and cultured in Mc COY’s 5a medium with 5% FCS for 24 h. The following day, the cells were stimulated in serum-free medium with 40 mM ciglitazone for 24 h. Supernatants were collected and TRAIL was quantified by enzyme-linked-immunosorbent assay (Diaclone). Tumor xenograft model Five to eight-week-old female nu/nu nude mice were maintained according to European Union guidelines for use of laboratory animals. In vivo experiments were performed in compliance with the French guidelines for experimental animal studies. Experi- mental protocols were approved by the Departmental Direction of the Veterinary Division of the Doubs, France (authorization certificate Nu25-27/Approval ID: B 25-056-7). Exponentially growing human bladder cancer cell lines RT4 and T24 were subcutaneously injected into the right flank of female nude mice as follows: 56106 cells in 50 mL PBS with 25% decomplemented FCS for both cell lines but with 50 mL Matrigel (BD Biosciences) for T24 cells. Animals were examined twice a week for tumor development. When tumor size reached 40 mm3 in volume, mice were randomly divided into control and treated groups containing ten mice each. Intraperitoneal injections of ciglitazone were References 1. Epstein JI, Amin MB, Reuter VR, Mostofi FK (1998) The World Health Organization/International Society of Urological Pathology consensus classifi- cation of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol 22: 1435–1448. 2. Lee R, Droller MJ (2000) The natural history of bladder cancer. Implications for therapy. Urol Clin North Am 27: 1–13, vii. 3. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, et al. (1995) An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 270: 12953–12956. 4. Day C (1999) Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med 16: 179–192. Ciglitazone Induces Bladder Tumor Cells Apoptosis weekly administered at a dose of 15 mg/kg during three weeks. Control animals received only saline vehicle following an identical schedule. Tumor size was monitored twice a week by measuring length and width with a calliper. Tumor volume was estimated using the following formula: volume = width26length6K. Mice were sacrificed one week after the third injection and tumors were removed. Immunohistochemistry Vehicle- and ciglitazone-treated tumors were collected, fixed in 4% formalin and paraffin-embedded. Then, blocks were cut serially at 5 mm thick, deparaffinized in toluene, rehydrated in graded ethanol. Antigen retrieval was achieved by micro-wave treatment in 0.5 M Tris-buffered saline (pH 6.0) at 750 W for 30 minutes. Sections were then incubated with primary antibodies at room temperature using an automated immunohistochemical processor (Techmate 500 Plus, DakoCytomation, Trappes, France) according to the manufacturer’s instructions. The biotinylated primary antibody anti-Ki-67 (DakoCytomation, MIB-1, 1:150) was used for evaluation of proliferating cells. Activated caspase 3 was detected with Apoptosis marker: SignalStain cleaved Caspase 3 (Asp175) IHC Detection kit (Cell Signaling). Slides were then treated with alkaline phosphatase- conjugated avidin. Finally, alkaline phosphatase activity was revealed by NBT-BCIP. Sections were counterstained with Harris’ hematoxylin, dehydrated through alcohol and mounted using a standard procedure. Negative controls were obtained by omitting the first antibody. Slides were then examined and pictures were taken using Zeiss Axioskop 40 photomicroscope. Statistical analyses For in vitro experiments, two-tailed unpaired Student’s t test was used to examine the significant differences between groups. Data are expressed as mean 6 SD of three independent experiments or as specified for each figure. The in vivo therapeutic efficacy of ciglitazone was assessed by evaluating the tumor volume development over time with two-way ANOVA test. Differences between control and treated mice at each post-graft time were determined by Student’s t test. Values with P,0.05 were considered significant. Acknowledgments We thank Sylviane Coumes-Marquet and Claude Collard for helpful advice for immunohistochemistry, Virginie Mougey for flow cytometry analysis and Franck Monnien for statistics. Author Contributions Conceived and designed the experiments: IL SF. Performed the experiments: M-LP SF IL. Analyzed the data: IL SF M-LP. Wrote the paper: IL SF M-LP HB. 5. Rumi MA, Ishihara S, Kazumori H, Kadowaki Y, Kinoshita Y (2004) Can PPAR gamma ligands be used in cancer therapy? Curr Med Chem Anti-Canc Agents 4: 465–477. 6. Chaffer CL, Thomas DM, Thompson EW, Williams ED (2006) PPARgamma- independent induction of growth arrest and apoptosis in prostate and bladder carcinoma. BMC Cancer 6: 53. 7. Guan Y, Zhang Y, Davis L, Breyer MD (1997) Expression of peroxisome proliferator-activated receptors in urinary tract of rabbits and humans. Am J Physiol 273: F1013–1022. 8. Nakashiro KI, Hayashi Y, Kita A, Tamatani T, Chlenski A, et al. (2001) Role of peroxisome proliferator-activated receptor gamma and its ligands in non- neoplastic and neoplastic human urothelial cells. Am J Pathol 159: 591–597. PLoS ONE | www.plosone.org 11 December 2011 | Volume 6 | Issue 12 | e28354