Fig 2: Caspase-8 is activated after HSV-1 infection and required for HSV-1-induced caspase-3 activation and apoptosis in MEFs.A Caspase-8 (IETDase) activity assay of total extracts of wild-type (wt) MEFs infected with 10 m.o.i. of HSV-1 for 0 to 36 h. Results are presented as relative fluorescence units (RFU). B Anti-caspase-8 western blot analysis of total extracts of MEFs infected with HSV-1 for 0, 8, 14 and 24 h, showing the inactive p55 caspase-8 pro-form and the cleaved, active p43/p41 and p18/p10 forms. The right line shows an extract from caspase-8−/− MEFs. C Percentage of FITC-Annexin-V/7-AAD-negative (surviving) wt and caspase-8−/− (Casp-8−/−) MEFs infected with HSV-1 for 0 (NT), 24, 48 and 72 h. D Anti-caspase-3 western blot analysis of total extracts of wt and caspase-8−/− MEFs infected with HSV-1 for 8, 14 and 24 h showing the inactive p32 caspase-3 pro-form and the cleaved, active p17 form. Data in A and C represent the means of 3–6 independent experiments ± SD. Statistical evaluation by one-way ANOVA: **p < 0.01; ***p < 0.001. In B and D, β-actin served as a loading control.

Fig 3: Caspase-8 is activated after HSV-1 infection and required for HSV-1-induced caspase-3 activation and apoptosis in U937 cells.A Caspase-8 (IETDase) activity assay of total extracts of wt U937 cells infected with 50 m.o.i. of HSV-1 for 0 to 36 h. B Caspase-3 (DEVDase) activity assay on total extracts of wt and caspase-8-/- (Casp-8-/-) U937 cells infected with HSV-1 for 0 (NT), 10 and 24 h. C Anti-caspase-8, -caspase-3 and -gD western blot analyses of total extracts of wt and caspase-8-/- U937 cells infected with HSV-1 for 0, 10 and 24 h showing the inactive p55 pro-form and the active p43/p41 and p18/p10 caspase-8 fragments, the active p20/p17 caspase-3 (Cl Casp-3) fragments and the expression of the viral gD protein. ß-Actin served as a loading control. D Percentage of FITC-Annexin-V/7-AAD-negative (surviving) wt and caspase-8-/- (Casp-8-/-) U937 cells infected with HSV-1 for 0 (NT), 10 and 24 h. Data in A, B and D represent the means of 3–7 independent experiments ± SD. Statistical evaluation by one-way ANOVA: **p < 0.01, ***p < 0.001.

Fig 4: Plk3 phosphorylates procaspase-8 at T273. (A) Recombinant GST-His-fused Plk3 (0.1 μg) was incubated with recombinant active His-tagged caspase-8-p18 and -p10 (10 U) at 30 °C for the indicated time periods. The reaction mix was blotted against Plk3 (BD), GST and caspase-8. (B) Schematic representation of the recombinant GST-fused procaspase-8 (GST-Casp8-WT), N-terminal part (GST-NT), C-terminal subdomains of procaspase-8 (GST-p18 and -p10) and its mutated forms used for in vitro kinase assays. (C) Recombinant GST-fused procaspase-8 and its subdomains were incubated with Plk3 for an in vitro kinase assay and visualized by autoradiography. (D) HEK 293T cells were transfected with Flag-tagged Plk3-WT and its mutants (T219D, K91R). The proteins were immunoprecipitated using anti-Flag M2 affinity gel. Recombinant GST-fused caspase-8-p18 was incubated with immunoprecipitated Plk3 for an in vitro kinase assay, analyzed by SDS-PAGE and stained with Coomassie (lower panel), and visualized by autoradiography (upper panel). (E) GST-Casp8-WT, NT, p18 and point mutants of p18, all verified by sequencing in both directions, were incubated with recombinant GST-His-fused Plk3 (0.1 μg) for an in vitro kinase assay, analyzed by SDS-PAGE, stained with Coomassie (lower panel), and visualized by autoradiography (upper panel). (F) Recombinant GST-fused p18-WT, p18 T273A and increasing amounts of Casp8-WT and Casp8 T273A were incubated with recombinant GST-His-fused Plk3 (0.1 μg) for an in vitro kinase assay, analyzed by SDS-PAGE, stained with Coomassie (lower panel), and visualized by autoradiography (upper panel).

Fig 5: Plk3 and procaspase-8 interact. (A) HEK 293T cells were co-transfected with GFP-Plk3 or GFP-Rip3 and Flag-procaspase-8. GFP-Rip3 was used as a positive control for the co-IP with procaspase-8. Precipitates were immunoblotted for Flag and GFP. (B) The binding ability of GST-fused procaspase-8 was analyzed in pull-down assays. GST-procaspase-8 or GST alone (as a control) was incubated with HeLa cell lysates. The pull-down was analyzed by immunoblotting for Plk3 and GST. (C) Schematic representation of recombinant GST-fused full-length Plk3 (GST-Plk3-WT) and truncated forms of Plk3 (GST-Plk3-NT1, -NT3 and -CT1) used in pull-down assays. (D) GST-Plk3-WT or Plk3 subdomains were incubated with HeLa cell lysates for pull-down assays. The immunoblot analysis was performed using caspase-8- and GST-antibodies. (E) HeLa cells were treated with 50 ng/ml nocodazole (Noc) for 16 h to enrich for mitotic cells. Endogenous caspase-8 was immunoprecipitated from cell lysates. Normal mouse IgG was used as a control. The immunoprecipitates and the lysates were immunoblotted for caspase-8, Plk1, Cdk1, Cyclin B1, Plk3 and Aurora B. (F) HeLa cells were treated with 50 ng/mL CD95L and CHX for 0 h and 2 h. Endogenous caspase-8 was immunoprecipitated from cell lysates with anti-caspase-8 p18 goat antibody. Normal goat IgG (Santa Cruz) was used as a control. The immunoprecipitates and the lysates were immunoblotted for Plk3, caspase-8, cleaved caspase-8 and p10. (G) The interaction of Plk3 and procaspase-8 (Casp8) was monitored via in situ PLA. HeLa cells were labeled with the anti-Plk3 (Abcam, rabbit) and anti-Casp8 (mouse) (left panel) or anti-Plk3 (BD, mouse) and anti-Casp8 (rabbit) (right panel) antibodies. Single antibody staining (Plk3 or Casp8) was used as a control. Scale bar: 10 µm. Each bar represents the mean value ± SD (n = 3). The differences between single and double antibody staining were statistically significant by Student's t-test (lower panels) (*P = 0.05; **P = 0.005).