Figure 2.

Apoptosis assays for 15d-PGJ₂ in three RCC cell lines. (A, B) Induction of chromatin condensation by 15d-PGJ₂. Typical fluorescence micrographs of Caki-2 cells stained with Hoechst 33342 (A) and percentages of chromatin-condensed cells (B). Cells were treated with 15d-PGJ₂ for 24 h at the IC₅₀ of each cell line and stained with Hoechst 33342 for 15 min at room temperature. They were then observed under a brightfield fluorescence microscope with UV excitation and the percentage of chromatin-condensed cells was determined. Data represent the mean ± S.D. from 4 independent preparations. Statistically significant difference from the control: **p < 0.01 assessed by t-test. (C) Effects of 15d-PGJ₂ on caspase-3 activity. Cells were seeded in 96-well plates at 5 × 10³ /well and cultured for 24 h. After exposure to 15d-PGJ₂ at the IC₅₀ of each cell line for 24 h, caspase-3 activity was assessed with a fluorimetric Caspase 3 Assay Kit according to the manufacturer's instructions. Enzymatic activities were determined as initial velocities corrected with the quantity of protein. Data represent the mean ± S.D. from 4 independent preparations. Statistically significant difference from the control: **p < 0.01 assessed by t-test. (D) Effects of a pan-caspase inhibitor on 15d-PGJ₂-induced cell death. Cells were precultured for 24 h at 5 × 10³ /well in 96-well plates and exposed to 15d-PGJ₂ with or without Z-VAD-FMK (100 μM) for 24 h. Cell viability was assessed by fluorescent assay, and data represent the mean ± S.D. from 4 to 5 independent preparations. Statistical significance was assessed by t-test.