Fig. 8. CP-31398-induced sequestration of several anti-apoptotic proteins in autophagosomes potentiates granzyme B-induced MOMP and caspase-3 cleavage.

a, b CP-31398 improves mitochondrial outer membrane permeabilization (MOMP) measured by Dioc6(3) staining in MDA-MB231 cells incubated with NK cells isolated from a healthy donor (NKd) at the E:T ratio of 5:1. c CP-31398 increases caspase-3 cleavage in MDA-MB231 cells incubated with NK cells isolated from a healthy donor (NKd) at the E:T ratio of 3:1. The normalized ratio of full length or cleaved caspase-3, -9 or PARP/Actin was calculated by densitometry analysis. d, e CP-31398 increases MOMP measured by Dioc6(3) staining (c) and caspases-9 and -3 cleavage (d) in MDA-MB231 cells treated 30 min with PFN/GzmB. f CP-31398 increases PFN/GzmB-induced apoptosis, measured by flow cytometry using M30 mAb staining (which recognizes a cytokeratin-18 epitope, revealed after effector caspase cleavage). Representative flow cytometry histograms are shown. Numbers indicate the percentage M30 positive cells (mean ± s.d. from three independent experiments). g PFN/GzmA-induced cell death, measured by Annexin-V staining, is unaltered following CP-31398 treatment. Data are representative of three independent experiments (a, c, e) or are the mean ± s.d. of three independent experiments (b, d, g). The p values (b, d, g) were determined by unpaired two-tailed Student's t test. NS non-significant