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. 2008 Oct 6;183(1):101–116. doi: 10.1083/jcb.200801099

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© 2008 Degtyarev et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 7. — Accumulation of AVOs preceded plasma membrane rupture and correlated with the appearance of apoptotic and anucleated cells with Akti-1/2 and CQ treatment. (A) PC3 cells treated with DMSO, 10 μM Akti-1/2, 10 μM CQ, or both under 5% FBS were followed for 3 d using time-lapse microscopy. Representative images of the cells at the indicated time points are shown. White arrowheads indicate the fusion between two adjacent cells before plasma membrane rupture in cells treated with both agents. Full time-lapse videos are available in Videos 1–4, available at http://www.jcb.org/cgi/content/full/jcb.200801099/DC1. Bar, 10 μm. (B) PC3 cells treated with the indicated agents were stained with AO and analyzed by multispectral imaging flow cytometry. (left) Brightfield (BF), nuclei (green), vacuoles (red), and green/red composite images of three representative cells with each treatment are shown. Bars, 10 μm. (middle) plotting AO green intensity versus AO green bright detail area revealed three distinct populations: R2 anucleated cells, R3 apoptotic cells, and R4 live cells. (right) AO red intensity for R4 is plotted on the histogram with an arbitrary gate (R5) drawn to include events with the brightest AO red intensity. R2, R3, and R4 histograms are overlaid in the Akti + CQ plot only. (C) Statistics for each population shown in B. *, percentage of total single cells; **, mean fluorescence intensity of R4 live cells; ***, percentage of R4 live cells.