Fig. 6

Extracellular ATP-induced changes in JfCaspase gene expression, enzymatic activity and apoptosis in Japanese flounder head kidney macrophages. a JfCaspase 2, 3, 6 and 8 gene expression changes following 1 mM ATP stimulation in the HKMs were measured by qRT-PCR with beta-actin as an internal reference gene. b The eATP-induced changes in JfCaspase 2, 3, 6 and 8 enzymatic activity in Japanese flounder HKMs were measured with the respective enzyme substrates (Ac-VDQQD-pNA, Ac-DEVD-pNA, Ac-VEID-pNA and Ac-IETD-pNA, respectively), and are presented as the pNA levels calculated from a standard curve. Asterisks (*) indicate significant differences in JfCaspase activity compared with that of the untreated control group (p < 0.05). All data are presented as the means ± standard deviation of triplicate determinations from one representative experiment. The other two independent experiments showed similar results. c eATP-induced changes in DNA fragmentation in the HKMs. Untreated HKMs served as controls to show intact genomic DNA. In a parallel experiment, HKMs were treated with 1 mM ATP for 12 or 24 h. After that, genomic DNA was extracted, and DNA fragments were assessed by 2% agarose gel electrophoresis and ethidium bromide staining. M: 100 bp DNA ladder (Fermentas). d Inhibition of caspase enzymatic activity inhibited eATP-induced DNA fragmentation in HKMs. HKMs were pre-incubated with or without 100 μM pan-caspase inhibitor Z-VAD-FMK for 30 min and then treated with 1 mM ATP for 12 or 24 h in the presence or absence of 100 μM Z-VAD-FMK. DNA fragments were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining