Nuclear condensation and fragmentation induced by GzmH. (A) Quantification of apoptotic nuclei after Hoechst 33342 staining 4 and 12 hours after the treatments indicated. The columns represent the mean with its standard deviations from triplicate measurements. (B) DNA content analysis of differentially treated K562 cells after 5, 12, and 24 hours. The pancaspase inhibitor zVAD-fmk was used at 100 μM. The 2 characteristic peaks (G₁ and G₂M, left and right peaks, respectively) of dividing cells were gradually lost, while cells with lower DNA content values in the so-called sub-G₁ area appeared (indicated by M₁). (C) Quantitative representation (n = 3; ± SD) of experiment B. Following the same trends as seen in the FACS data, GzmB-treated cells show early DNA degradation while GzmH works a slow but progressive diminution of DNA, with the percentage of sub-G₁ cells matching that induced by GzmB at the 24-hour time point. (D) GzmH treatment does not cause apoptotic DNA laddering. K562 cells were treated for 4 and 12 hours as indicated, and cellular DNA was analyzed on a 1% agarose gel. The experiment was repeated 3 times.