Ciglitazone Induces Bladder Tumor Cells Apoptosis Figure 6. Ciglitazone lets TRAIL-resistant T24 cells to respond to TRAIL through caspases activation and c-FLIP and survivin downregulation. T24 cells were treated or not with 40 mM ciglitazone for 24 h or human recombinant TRAIL (50 ng/ml) for 12 h or cotreated with ciglitazone and TRAIL (added here for the last 12 h). (A, top) The percentage of cells showing hypodiploid DNA content (sub-G1 peak) was evaluated by flow cytometry analysis. 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 individual treatment-exposed cells with the use of two-tailed unpaired Student’s t test; (A, bottom) Immunoblotting detection of caspases 8, 9 and 3, PARP and Bid. (B) Cells were preincubated 1 h with 50 mM caspase 8 (Z- IETD-FMK) or 9 (Z-LEHD-FMK) specific inhibitor before indicated treatment; top, the percentage of cells showing hypodiploid DNA content was evaluated by flow cytometry analysis; bottom, caspases 8, 9, 3 and Bid cleavage was assayed by western-blotting analysis. (C) Cells were preincubated with or without monoclonal antibodies blocking DR4 and DR5 receptors for 1 h and stimulated as indicated. The percentage of cells showing hypodiploid DNA content was evaluated by flow cytometry analysis. (D) Immunoblotting detection of c-FLIP and survivin from T24 whole cell lysates. PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28354