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. 2004 Jun;78(12):6543–6555. doi: 10.1128/JVI.78.12.6543-6555.2004

FIG.1.

FIG.1.

Caspase activation in lyssavirus-infected neuroblastoma cells. Cells were infected with THA, MOK, or LAG at an MOI of 1. After incubation at 37°C for the indicated times, cells were processed. Results are expressed as means of data obtained in three independent experiments. Error bars, standard deviations. Significant effects are indicated by asterisks, pound signs, and plus signs (P < 0.05). (A) Percentages of lyssavirus-infected cells undergoing apoptosis and cell death according to the TUNEL assay and the amount of LDH released. (B) Caspase-1, -2, and -3 activity assays were evaluated by using Ac-DEVD-AMC-, VDVAD-pNA-, and Ac-YVAD-AMC-labeled substrates, respectively. (C) Caspase-6 activity was quantified at the single-cell level by using a medium containing a specific labeled peptide inhibitor (FAM-VEID-fmk) that recognizes the activated form of caspase-6. (D) Caspase-8 activity was quantified at the single-cell level by using a medium containing a specific labeled peptide inhibitor (FAM-LETD-fmk) that recognizes the activated form of caspase-8. LETDase activities were quantified in neuroblastoma cells infected at MOIs of 1 and 30. (E) Caspase-10 activity was quantified at the single-cell level by using a medium containing a specific labeled peptide inhibitor (FAM-AEVD-fmk) that recognizes the activated form of caspase-10. AEVDase activity was quantified in infected neuroblastoma cells (MOI, 30). (F) Western blot analysis of caspase-8 revealed the p54 fragment of the precursor form of caspase-8 (Pro casp-8). β-Actin was included as a loading control. NEG, negative control. (G) Caspase-9 activity was quantified at the single-cell level by using a medium containing a specific labeled peptide inhibitor (FAM-LEHD-fmk) that recognizes the activated form of caspase-9. LEHDase activities were quantified in neuroblastoma cells infected at MOIs of 1 and 30. (H) Cells were incubated in the presence or absence of a cell-permeant, irreversible pan-caspase inhibitor, Z-VAD-fmk, and a specific caspase-8 inhibitor, Z-IETD-fmk. CONTR, control.