Caspase biosensors are activated by perforin-dependent and -independent mechanisms. The PRF dependence of the biosensor signal was tested with shRNA silencing of PRF1 in KHYG1 cells (A-D) and a patient-derived line deficient in PRF (E-H). (A) Expression of PRF in parental KHYG1 line and in KHYG1 shPRF transduced with shRNA specific to PRF1 3′UTR. PRF expression relative to KHYG1 is shown. Relative PRF expression to KHYG1 is shown below. (B) Cytotoxicity of KHYG1 shPRF cells against K562 targets is decreased. Error bars represent SEM from >10 independent experiments. Luciferase biosensors were sensitive to PRF knockdown in KHYG1 shPRF cells after 1:1 conjugation with K562 cells expressing (C) GLS.VGPD or (D) GLS.IETD. (E-H) HVS-CL line from PRF^−/− patient or PRF^+/+ donor reveals late caspase activation after 1:1 conjugation with K562 expressing: (E) GLS.VGPD, (F) GLS.IEAD, (G) GLS.IETD, or (H) GLS.DEVD. For luciferase assays, each datum point represents the mean of 4 replicate wells. One representative graph is shown from 3 independent experiments with similar results.