Figure 5

BIRD-2-induced Ca²⁺ elevation and Ca²⁺-mediated caspase activation. (a) Shown are continuous single-cell recordings of cytoplasmic Ca²⁺ by digital imaging, performed simultaneously in 65 H2171 SCLC cells following addition of 10 μM BIRD-2 (arrow). Traces represent Ca²⁺ levels in individual cells. (b) Same as panel (a) except in Scr-treated H2171cells. (c) Bar graph represents the percentage of SCLC, NSCLC, and normal lung epithelial cell lines responding to BIRD-2 with a significant elevation of cytoplasmic Ca²⁺ above basal Ca²⁺ levels. Cytoplasmic Ca²⁺ was measured as in panel (a). This figure summarizes a single experiment analyzing >80 cells in each cell line; shown is the average peak cytoplasmic Ca²⁺ elevation in each cell line within 30 min following addition of 5 μM BIRD-2. (d) Cytoplasmic Ca²⁺ was recorded as in panel (a) except that H2171 cells were pretreated 1 h before adding 10 μM BIRD-2 with 5 μM BAPTA-AM to chelate intracellular Ca²⁺ and thus block cytoplasmic Ca²⁺ elevation. (e) Shown is a bar graph representing the peak cytoplasmic Ca²⁺ elevation (mean±S.D., three experiments) induced by 10 μM BIRD-2 treatment, with or without 10 μM BAPTA-AM pretreatment. Results indicate that BAPTA-AM pretreatment inhibits BIRD-2-induced Ca²⁺ elevation. (f) BAPTA-AM pretreatment decreases BIRD-2-mediated caspase 3/7 induction, measured by IncuCyteZOOM as in Figure 3c. Experiments in panels d–f were performed at least three times, using 2–5 μM BAPTA-AM and 15–20 μM BIRD-2. Error bars in panel (e) represent mean±S.D., N=85 cells. **P<0.01