Fig. 2. Visualizing externalized PIPs on apoptotic cells.

A Representative images of externalized PIPs on apoptotic HeLa cells. HeLa cell apoptosis was induced by 16 h treatment with either an anti-FAS antibody (100 ng/ml), camptothecin (10 μM) or cycloheximide (100 μM). Cells were stained using an anti-PI(3,4,5)P₃ antibody, recombinant MYC-tagged PLCδ PHD, or AKT-PHD protein. All were visualized using an FITC-labeled secondary antibody and DAPI (scale bars, 10 μm). Inserts use differential interference contrast microscopy. B Representative images of externalized PI(3,4)P₂ and PI(3,4,5)P₃ detection using a recombinant eGFP/AKT PHD fusion protein on CHO cells expressing mCherry/AKT PHD fusion protein. Apoptosis was induced in CHO cells that stably expressed the mCherry/AKT PHD fusion protein to visualize intracellular PI(3,4)P₂ and PI(3,4,5)P₃, while externalized PIPs were visualized using recombinant eGFP/AKT PHD fusion protein (scale bars, 10 μm). Three-dimensional reconstructed images were made using Zeiss Zen SP2 software. Camptothecin-treated cells typically displayed either a large bleb or small patches of eGFP-positive staining; examples of both are shown. Supplementary Movie 1 provides a time-lapse video. See also Supplementary Figs. 2–4.