The Amazing Innovative TRIB1 Tactic Invented By My Buddy

For P. pouchetii cultures infected with PpV-01B virus, DNA fragmentation appeared as a distinct laddering phenotype (Fig.?3B, black arrows). Genomic DNA fragmentation selleck products in PpV-infected cultures of P. pouchetii was already apparent in samples taken immediately after PpV addition at the start of the experiment (0 h, Fig.?3B). With time allowances for centrifugation and resuspension in lysis buffer, this would indicate that DNA fragmentation in P. pouchetii was induced within 30 min after virus addition. The same results were observed for at least two independent experiments for both H. ericina and P. pouchetii. We observed DNA laddering patterns for H. ericina and P. pouchetii cultures upon treatment with camptothecin (Supplementary data, Fig. S1), a DNA topoisomerase I inhibitor known to induce replication arrest and PCD (Sen et al., 2004), thereby confirming the inducible PCD-like DNA fragmentation phenotype associated with virus infection. Similar DNA fragmentation was not observed after 24 h for untreated control cultures (data not shown). Fig.?3. Induction of DNA fragmentation during CeV infection of Haptolina ericina (A) and PpV infection of Phaeocystis pouchetii (B) cultures. Lane identifiers: TRIB1 M, dsDNA molecular weight marker with sizes shown in basepairs; A, positive control mammalian U937 ... IETDase catalytic activity in cell extracts We observed a dramatic increase in IETD-AMC cleavage only in virus-infected cultures (Fig.?4, black symbols) for both phytoplankton. The ratio of click here IETDase catalytic activity in CeV-infected H. ericina cultures increased from 3-fold at 48 h post-virus addition to 20-fold by 72 h post-virus addition (Fig.?4A, grey dashed line) compared with control cultures. Relative IETDase specific catalytic activity increased from a 1.5-fold difference between P. pouchetii, control and PpV-infected cells at 36 h post-virus addition to a 4-fold difference at 72 h post-virus addition (Fig.?4B, grey dashed line). Fig.?4. Cleavage of the fluorogenic caspase-8 substrate isoleucyl-glutamyl-threonyl-aspartic acid-7-amino-4-methylcoumarin (IETD-AMC) in soluble cell extracts of control (open circles) and virus-infected (black circles) cultures of H. ericina (top) and P. pouchetii ... Pre-treatment of H. ericina and P. pouchetii cell extracts with z-VAD-fmk for 1 h prior to addition of IETD-AMC confirmed both the biochemical activity and its specificity in these phytoplankton species. The presence of the z-VAD-fmk inhibitor resulted in a 60�C80% reduction in IETDase catalytic activity in both control and virus-infected cell extracts (two-tailed t-test, all P