Fig. 3. Contribution of the apoptotic pathway to cytotrophoblast cell death. All cells were cultured for 8 hours at 2% O₂ in medium supplemented as described below and in the Materials and methods. (A) Internucleosomal cleavage of DNA was assessed by agarose gel electrophoresis and ethidium bromide staining of genomic DNA. Cells were cultured in medium containing vehicle (lane 1), CRM197 (lane 2), anti-HBEGF (lane 3), or antibodies against both HER1 and HER4 (lane 4). (B) Cells exposed to CRM197 were dually stained using DAPI and the TUNEL method. Pyknotic nuclei visualized by fluorescence microscopy in the upper panel were also positive for TUNEL (arrows). (C) Annexin V binding to phosphatidylserine exposed on the surface of apoptotic cells is nearly absent in control cells (lower left panel) compared with cells treated with CRM197 (lower right panel). The upper panels show the same fields labeled with DAPI to indicate the relative cell densities. (D) Cell death was quantified by TUNEL assay after culture in medium containing CRM197. The indicated caspase inhibitors (2 µg/ml) or an inactive analog (200 µg/ml) were also added to the medium. Specificity of each compound is shown in parentheses. The dashed line indicates the level of TUNEL in cells cultured without CRM197. ^*P<0.05, compared with vehicle control.