Figure 3.

Effect of KICA on PARP cleavage and caspase activity. (A) PARP cleavage in C6 cells assessed by Western blot analysis after treatment with DMEM/0.5% FCS containing 25 mM KICA for 0 (control) 1, 2, or 3 h as indicated. The amount of cleaved PARP was defined by measuring the signal intensity of the M_r 85,000 (85K) cleavage product expressed as a percentage of the total combined signal for the 85K and M_r 116,000 (116K) bands. (B) Caspase activation in C6 cells after treatment for 3 h with DMEM/0.5% FCS alone (con) or in the presence of 50 mM KICA (black bar) or 1 μM SSP (gray bar) as indicated. Caspase activation was assessed by colorimetric assay using DEVD-pNA as a substrate. Results were normalized for differences in protein content between lysates, and the results are expressed as fold increase ± SEM in caspase activity relative to untreated cultures (n = 6). (C) Effect of caspase inhibitors on the toxic effects of KICA. C6 cells were incubated for 20 h with 10% conditioned medium (10% CM), 50 mM KICA, or 1 μM SSP either alone (white bars) or in the presence of 100 μg/ml BAF (black bars), DEVD-FMK (gray bars), or IETD-FMK (hatched bars). Cell death was then assessed by MTT assay.