Ciglitazone Induces Bladder Tumor Cells Apoptosis Figure 2. Ciglitazone induces apoptosis in T24 cells. Cells were exposed for 24 h to vehicle or ciglitazone at the indicated concentrations. (A) The percentage of cells showing the hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis. (B), (C) The cleavage of caspases 9, 8, 3 and PARP as well as the expression of pro- and anti-apoptotic proteins were determined by western blotting analysis. (D, left) Cells were preincubated 1 h with 50 mM caspase 8 (Z-IETD-FMK) or 9 (Z-LEHD-FMK) specific inhibitor before treatment with 40 mM ciglitazone for 12 h and assessment of caspases 8, 9, 3 and Bid processing was analysed by western-blotting. b-actin was used as an internal loading control ; (D, right) After indicated treatments, cells were stained with PI for DNA fragmentation analysis by flow cytometry. Data are means 6 SD of 3 independent experiments performed in triplicates. *P,0.05, significant differences compared with untreated cells ; **P,0.05, significant differences compared with ciglitazone-treated cells with the use of two-tailed unpaired Student’s t test. doi:10.1371/journal.pone.0028354.g002 tumors were formed, mice were treated by vehicle or ciglitazone (15 mg/kg) at the rate of one injection per week for three weeks. Tumor volume was measured twice per week until the mice were sacrificed. The tumor incidence was 100% in all animals. The volume of the tumors in T24 cells-grafted mice did not increase in ciglitazone-treated animals compared to vehicle-treated control animals. There was a significant difference from the third injection of ciglitazone (P,0.05) (Figure 5A, left panel). In RT4 cells-grafted mice, tumor volume seemed to develop less than in control mice upon ciglitazone treatment but this was not statistically significant (Figure 5A, right panel). To determine whether the inhibition of the tumor development by ciglitazone is due to inhibition of proliferation, apoptosis, or both, we evaluated Ki67 expression and caspase 3 activation on tumor tissue sections by immunohis- tochemical analysis. The number of Ki67-positive cells was lower in T24 and RT4 cells-xenografted tumor sections from ciglita- zone-treated animals compared to tumor sections from untreated animals. On the contrary, there was no detectable activated caspase 3 in RT4 cells-xenografted tumor sections and a strong activated caspase 3 staining in T24 cells-xenografted tumor sections from ciglitazone treated animals compared to tumor sections from untreated animals (Figure 5B). These results PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28354