Caspase-10 in Fas Signalling Figure 6. zVAD-fmk does not abrogate over-expressed caspase-10-triggered cell death in HeLa cells. A, Total protein extracts derived from wild-type Jurkat (A3) and HeLa cells were analysed by Western blot using anti-caspase-8, anti-caspase-10 or anti b-actin antibodies. B–D, HeLa cells were transfected with plasmid encoding EGFP, EGFP-tagged wild-type caspase-8 (Casp8) or -10 (Casp10). B, Cells were analysed by flow cytometry 16 hours post-transfection. Values indicated are percentages of EGFP-expressing cells. C, Immediately after transfection, 40 mM zVAD-fmk was added or not to the cell culture medium and cells were further incubated for 24 hours. During the last 8 hours, cells were incubated with or without 1 mg/mL FasL. Protein extracts were analysed by Western blot using the indicated antibodies. Cleaved PARP expression was analysed at low (up panel) and high (low panel) exposure. (*: EGFP-tagged pro-caspase-8 or 10, **: EGFP-tagged small catalytic caspase-8 or -10 subunit). The <26 kDa band obtained in caspase-8 and -10 expressing cells remains to be characterized. D, HeLa cells were transfected and cultured for 24 hours in the presence or absence of zVAD-fmk. Cells were labelled with annexin-V/APC. Annexin-V binding analysis was restricted to the EGFP-expressing cells. Data are means 6 SEM of five independent experiments. ***p,0.001; * p,0.05. doi:10.1371/journal.pone.0013638.g006 completely abrogated by zVAD-fmk in caspase-10-expressing cells (Figs 8A and B). Even at a concentration of 100 mM, zVAD-fmk failed to abolish cell death in Casp10+ cells (data not shown). Thus, under our experimental conditions, whereas zVAD-fmk efficiently prevented FasL-induced apoptosis in caspase-10-defi- cient HeLa cells, moderate expression of exogenous caspase-10 overcame, to some extent, the resistance conferred by zVAD-fmk. Western blot analysis further indicated that, in caspase-10-null cells, FasL triggered endogenous caspase activation, as illustrated by the decrease of pro-caspase-8 and PARP cleavage (Fig. 8C). In contrast, FasL promoted no or little decrease of pro-caspase-8 in caspase-10-expressing HeLa cells. However, EGFP-tagged cas- pase-10 was efficiently activated as illustrated by the complete loss of pro-caspase-10 expression and the appearance of the 40 kDa band, similar to that observed in transiently transfected cells (see Fig. 6C). PARP was also efficiently cleaved, despite weak endogenous caspase-8 activation (Fig. 8C). This last observation may indicate that EGFP-tagged caspase-10 competes with endogenous caspase-8 to interact with FADD, therefore limiting the recruitment and activation of endogenous caspase-8 at the DISC level. Interestingly, whereas zVAD-fmk efficiently blocked endogenous caspase-8 and PARP processing, it had no or little effect on EGFP-tagged caspase-10 cleavage (Fig. 8C). Altogether, our data demonstrate that zVAD-fmk cannot potently prevent caspase-10 processing in Fas signalling, enabling the activation of a caspase-10-dependent alternative cytotoxic signalling pathway in response to FasL. Discussion The present observations indicate that the initiator caspase-10 is involved in FasL-induced cell death and the broad-spectrum pancaspase inhibitor zVAD-fmk fails to abrogate caspase-10 activity. Indeed, cell death triggered by FasL was fully impaired in I9-2e (caspase-8 and -10-doubly deficient), but not in I9-2d (caspase-8-deficient but caspase-10-proficient) Jurkat cells. Cas- pase-10 expression in I9-2e cells restored sensitivity to FasL. Moreover, zVAD-fmk did not abrogate FasL-induced toxicity in PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13638