Lack of homing endonuclease toxicity.

<p>(<b>a</b>) Immunofluorescence images of cells stained for γ-H2AX. Nuclei were counterstained with DAPI. At least 330 cells were scored for the presence of foci under a 40X objective. (<b>b</b>) Live/dead analysis of cells 35 dpt. Left column, flow cytometric analysis for reporter GFP fluorescence and HE expression (mCherry fluorescence). The histograms from the middle (mCherry⁺ cells only) and right (mCherry⁻ cells only) columns show the results of the live/dead analysis; dead cells with compromised membranes stain with the amine-reactive fluorescent dye (Pacific blue). Positive control cells treated overnight with 1 µM staurosporine (STS, a cell death inducer) are shown in the bottom row. (<b>c</b>) Flow cytometry analysis for activated caspase-3. T1H4S cells were transduced at the indicated moi with lentiviral vector expressing either active Y2 I-<i>Ani</i>I or inactive E148D I-<i>Ani</i>I enzyme. At 3 dpt, cells were split into duplicate wells and assayed for cell death. One well for each condition was used for flow cytometric analysis of reporter GFP fluorescence and HE expression (mCherry fluorescence) after 6 h treatment with MG132. The second well of cells was used to assay for apoptosis by caspase-3 staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016825#s4" target="_blank">Material & Methods</a>. The positive control for apoptosis was obtained by treating T1H4S cells left unexposed to HE-expressing lentivirus with 1 µM staurosporine STS overnight. The left column shows the GFP and mCherry fluorescence analysis, and the middle (total cell population) and right (mCherry⁺ cells only) columns present the results of the caspase-3 analysis for each condition. (<b>d</b>) Cell cycle analysis of T1H4S cells transduced with lentiviral vector expressing either active Y2 I-<i>Ani</i>I or inactive E148D I-<i>Ani</i>I enzyme for 6 days, sorted for mCherry expression, and kept for 22 more days in culture. Cells were not treated with MG132 prior to staining and flow cytometry analysis. Blue, mCherry⁻ cells; red mCherry⁺. Arrow indicates the increased delay in G2 among cells exposed to Y2 I-<i>Ani</i>I.