Fig. 4. (A) Fluorescent TUNEL stain of thymus 2 days after 6 Gy irradiation. Apoptotic cells are labeled in green, and cell nuclei are stained with DAPI. parp ^+/+ are sparsely stained whereas parp-1^–/– and parp-2^–/– show a strong green staining of apoptotic cells. (B) PARP-1 and PARP-2 cleavage kinetics after 100 µM VP16 treatment in HL-60 cells. Z-VAD-fmk inhibitor was used at 0.5 µg/µl and was added 1 h before VP16 treatment. For western blot, 10⁵ cells were harvested at the indicated time, mixed with loading buffer and sonicated. Polyclonal antibodies anti-PARP-1 and anti-PARP-2 were used. (C) In vitro cleavage of purified mPARP-2 (2.5 µg) by active caspase-3 (8 U, Chemicon International) shows the same cleavage pattern in HL-60 cells after VP-16 treatment. The N-terminus of the 55 kDa band was micro sequenced and was found to be ⁵⁸DNRD⁶¹, a consensus cleavage site for caspases 3/7.