GzmH triggers mitochondrial depolarization and ROS. (A) GzmH induces a loss of the mitochondrial membrane potential ΔΨ_m. K562 cells were treated with GzmH or GzmB in the presence or absence of the pancaspase inhibitor for 4 or 24 hours and analyzed by flow cytometry after JC-1 staining. The loss of ΔΨ_m is detected by a decrease of red fluorescence (FL2) and has been quantified by the percentage of cells appearing in the R2 region. (B) Accumulation of cells with loss of ΔΨ_m over time. The experiment shows the mean of triplicates (± SD) and is representative of 2 experiments. Depolarization in most cells was already observed at the 4-hour time point after GzmB treatment; comparable effects were visible after 8 hours in GzmH-treated cells. (C) ROS levels measured by DHE staining in GzmH- and GzmB-treated K562 cells after 30 minutes and over the first 90 minutes. K562 cells were treated with the indicated granzymes and SLO^C530A, a mutated SLO variant that needs no prior reductive activation. The dye is oxidized by ROS to the strongly red fluorescent ethidium (R2 in the dot blots). H₂O₂ (1%) was used as positive control. (D) GzmH induced a rapid but transient ROS increase that peaked after 30 minutes. The data represent the average percentage of cells in area R2 of the raw FACS data. Shown is the mean of triplicates ± SD of 1 experiment representative of 2 experiments.