Figure 2

Investigating the importance of calcium signaling pathways for apoptosis. (A) Increases in Ca²⁺_(i) evaluated by the fluorescent calcium indicator FLUO-4 after addition of the bioprobe set. Data in this and later figures are presented as box plots (median (25 – 75^th percentile; 10^th – 90^th percentile)); (B) HCT116 cells were treated with the bioprobe set for 24 hours in the presence or absence of 10 μM BAPTA-AM. The generation of the apoptosis-specific caspase-cleavage product CK18-Asp396 was assessed using the M30-Apoptosense^® ELISA assay [15] and the degree of inhibition of caspase-cleavage was calculated for each compound; (C) Ca²⁺ is required during the late phase of apoptosis. BAPTA-AM was added at different times after addition of the bioprobe set and apoptosis was assessed at 24 hours of drug treatment; (D) Inhibiton of apoptosis by pre-treatment with ryanodine (10 μM) or dantrolene (50 μM), (E; F) BAPTA-AM blocks drug-induced modulation of the conformation of Bak. (E) HCT116 cells were exposed to 8 different drugs in the presence or absence of BAPTA-AM. All 8 drugs induced FLUO-4 fluorescence and BAPTA-AM sensitive apoptosis. Fixed cells were stained with an antibody that recognizes an N-terminal Bak epitope exposed after conformational activation during apoptosis [18, 43]. (F) Inhibition of drug-induced caspase-3 activation and Bak conformation by BAPTA-AM. Cells were exposed to one of the bioprobes (NSC106408), fixed and stained by an anti-Bak antibody and an antibody to active caspase-3.