Figure 2

Detachment from ECM induced apoptosis in normal epithelial MDCK and HBE cells. A, DNA laddering analysis of cell death. Normal cells and tumor cells from regular cell culture dishes (1 × 10⁷ ) were plated into 100-mm polyHEMA-coated dishes for 24 h and harvested. Cytosolic DNA was extracted and analyzed by agarose gel electrophoresis. B and C Annexin V staining analysis of cell death. 5 × 10⁵ MDCK, HBE, and H460 cells from regular cell culture dishes were plated into 60-mm polyHEMA-coated dishes for 24 h, harvested, fixed, and assessed by Annexin V/propidium iodide (AV/PI) DNA staining and flow cytometry. B, Percent of early apoptotic cells determined by flow cytometry using AV/PI binding. Quadrant D1: PI/AV +/-, necrotic; D2: PI/AV +/+, late apoptotic/necrotic; D3: PI/AV -/-, live cells; D4: PI/AV -/+, early apoptotic cells. C, Histograms of flow cytometry data. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. D, Immunoblotting analysis of cleaved caspase-3. After the indicated time periods cells were lysed and total protein was extracted separated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. "***": P < 0.001.