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. 2011 Dec 12;6(12):e28354. doi: 10.1371/journal.pone.0028354

Figure 6. Ciglitazone lets TRAIL-resistant T24 cells to respond to TRAIL through caspases activation and c-FLIP and survivin downregulation.

Figure 6

T24 cells were treated or not with 40 µM 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 ± 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 µM 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. (E) Cells were pretreated with 25 µM proteasome inhibitor MG132 for 30 min and stimulated with 40 µM ciglitazone for 12 h ; top, the percentage of cells showing hypodiploid DNA content was evaluated by flow cytometry analysis ; bottom, immunoblotting detection of c-FLIP and survivin. β-actin was used as an internal loading control. (B, C, E) Data are means ± SD of 2 independent experiments performed in triplicates. *P<0.05, significant differences compared with untreated cells ; **, P<0.05, significant differences compared with ciglitazone and TRAIL cotreated cells with the use of two-tailed unpaired Student's t test.