Figure 7.

YVADase and DEVDase activity measurements after C6 ceramide treatments. (A) Substrate specificity of C6 ceramide-induced caspase-1 (YVADase) and-3 (DEVDase) like activities in rice protoplast extracts. Caspase-like activities were assayed by measuring the fluorescence intensity of the cleaved specific caspase-1 substrate Ac-YVAD-AMC and caspase-3 substrate Ac-DEVD-AMC. The assay was repeated three times with similar results. Letters indicate that values are different based on (p < 0.05). Bars show standard deviations. (B) Effect of different protease inhibitors on DEVDase activity after C6 ceramide treatment for 6 h. The fluorescence units were measured and are expressed as percentage of the enzyme activity of solvent-treated protoplasts (100%). Protoplast extracts were pre-incubated with 50 μM Ac-DEVD-CHO, 200 μM pepstatin A, or 100 μM leupeptin for 1 h before adding Ac-DEVD-AMC. Error bars indicate ± SE from three technical replicates. The assay was repeated two times using independent samples. Asterisks show a significant difference from the enzyme activity of solvent-treated protoplasts Student’s t-test (*p < 0.05). (C, D) Effect of caspase-specific inhibitors on cell viability. Ac-YVAD-CHO (C) and Ac-DEVD-CHO (D) partially decrease the C6 ceramide-induced cell death. Protoplasts were pretreated with 20 μM caspase specific inhibitors Ac-YVAD-CHO or Ac-DEVD-CHO for 1 h and then treated with 100 μM C6 ceramide for 24 or 48 h. Degree of cell death was estimated by FDA staining. The assay was repeated three times with similar results. Letters indicate that values are different based on PLSD (p < 0.05). Bars show standard deviations.