FIG. 3.

Involvement of caspases in Daxx silencing-mediated apoptosis. (A) TUNEL assay showing synergistic induction of apoptosis by siDaxx and UV. HeLa cells were transfected with siDaxx or control reagent for 48 h. The caspase inhibitor z-VAD-fmk (z-VAD, 20 μM) was added 1 h prior to UV irradiation (50 J/m²). Apoptosis was determined by TUNEL (red) and DAPI (blue) staining 8 h after UV irradiation. (B) Effect of various caspase inhibitors on siDaxx-mediated apoptosis. HeLa cells were transfected with siC (lanes 1) or siDaxx (lanes 2 to 6), followed by treatment with actinomycin D (AD, 100 nM), UV irradiation (50 J/m²), or TNF-α (10 ng/ml) in the absence (lanes 2) or presence (lanes 3 to 6) of various caspase inhibitors. Cells were treated with the general caspase inhibitor z-VAD-fmk (lanes 3), the caspase 1 inhibitor z-YVAD-fmk (lanes 4), the caspase 8 inhibitor z-IETD-fmk (lanes 5), or the caspase 9 inhibitor z-LEHD-fmk (lanes 6). Apoptotic cells were detected by TUNEL assay and counting at least 200 cells in three different fields. The general caspase inhibitor z-VAD-fmk completely blocked siDaxx-enhanced apoptosis, while z-IETD-fmk and z-LEHD-fmk partially suppressed siDaxx-enhanced apoptosis.