Effects of knockdown of TC-NER components on convergent transcription-induced cell death.

<p>(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; XPA-1, 55%; XPA-2, 63%; CSB-1, 52%; CSB-2, 51%; ERCC1-1, 61%; ERCC1-2, 53%; XPG-1, 51%; XPG-2, 57%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; XPA-1, 33%; XPA-2, 31%; CSB-1, 29%; CSB-2, 26%; ERCC1-1, 39%; ERCC1-2, 31%; XPG-1, 25%; XPG-2, 40%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequencies of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *<i>P</i><0.05; **<i>P</i><0.001; ***<i>P</i><0.0001.</p

Percentage change in cell death in DIT7 and DIT7-R103 cells due to treatment with specific siRNAs.

a<p>The percentage increase in cell death due to treatment with a specific siRNA was calculated relative to the vimentin siRNA control as {[(% dead cells after specific siRNA)-(% dead cells after vimentin siRNA)]/(% dead cells after vimentin siRNA)}(100%). The data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046807#pone-0046807-g002" target="_blank">Figures 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046807#pone-0046807-g004" target="_blank">4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046807#pone-0046807-g005" target="_blank">5</a>.</p

Structure of the HPRT minigenes in DIT7 and DIT7-R103 cells.

<p>DIT7 cells carry a CAG₉₅ repeat tract and DIT7-R103 cells, which were derived from DIT7 cells by contraction of the repeat, carry a CAG₁₅ repeat tract. In both cell lines, the CAG tract is centered in the 2.1-kb intron in the single, randomly integrated <i>HPRT</i> minigene. The CAG repeat is about 1.6 kb downstream of the sense promoter and about 2.5 kb upstream of the antisense promoter.</p

Effects of MSH2 knockdown on convergent transcription-induced cell death.

<p>(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; MSH2-1, 41%; MSH2-2, 42%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; MSH2-1, 4%; MSH2-2, 9%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequency of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *<i>P</i><0.05; **<i>P</i><0.001; ***<i>P</i><0.0001.</p

Effects of knockdowns of MSH2, XPA and RNase H on convergent transcription-induced CAG repeat contraction.

<p>Contraction frequencies were calculated as the number of HPRT⁺ colonies divided by the number of viable cells, averaged over at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *<i>P</i><0.05; **<i>P</i><0.01.</p

Effects of RNase H knockdown on convergent transcription-induced cell death.

<p>(A) siRNA knockdowns in DIT7 cells. Frequencies of cell death are: vimentin, 47%; RNase H1-1, 49%; RNase H2A-1, 46%; RNase H1-1 plus RNase H2A-1, 54%. (B) siRNA knockdowns in DIT7-R103 cells. Frequencies of cell death are: vimentin, 22%; RNase H1-1, 27%; RNase H2A-1, 28%; RNase H1-1 plus RNase H2A-1, 44%. Frequency of cell death was calculated as the number of nonadherent cells divided by the sum of adherent and nonadherent cells. Data are the average frequency of cell death from at least 6 independent siRNA knockdown experiments. Error bars indicate standard deviations. Statistical significance relative to the vimentin control is indicated: *<i>P</i><0.05; **<i>P</i><0.001; ***<i>P</i><0.0001.</p

Doxycycline-induced transcription of the GFP gene.

<p><b>A</b>. Quantification of doxycycline-induced GFP transcription in GFP(CAG)₈₉ and GFP(CAG)₀ cell lines. Cells were induced with doxycycline for three days prior to analysis. Two independent primer sets, both specific for GFP exon 2, were used to amplify GFP transcripts by quantitative RT-PCR. For each primer set, the individual results were internally normalized to β-actin and then to the levels of GFP transcripts in uninduced GFP(CAG)₀ cells. The values for the uninduced levels of GFP transcripts in GFP(CAG)₈₉ cells are indicated. The increase over uninduced levels in GFP(CAG)₈₉ cells was 76-fold for primer set 1 and 98-fold for primer set 2. <b>B</b>. Transcription-induced changes in numbers of GFP+ cells in GFP(CAG)₈₉ cells. Cells were treated with doxycycline for 0, 24, or 48 hours prior to analysis for GFP+ cells by flow cytometry, using the High gate indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113952#pone-0113952-g004" target="_blank">Figure 4A</a> to define the population of GFP+ cells. The frequencies of GFP+ cells at 0, 24, and 48 hours, respectively, were 0.02±0.013%, 0.20±0.02%, and 0.37±0.01%, as determined by counting three samples of 50,000 cells. C. Kinetics of induction of GFP expression in GFP(CAG)₀ cells. Doxycycline (2 µg/mL) was added at time 0 to wells of 6-well plates containing 100,000 GFP(CAG)₀ cells. Individual wells were harvested at the indicated times and analyzed by flow cytometry, using the High gate indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113952#pone-0113952-g004" target="_blank">Figure 4A</a> to define the population of GFP+ cells.</p

GFP fluorescence in GFP(CAG)₈₉ cells and in GFP(CAG)₀ cells before and after addition of doxycycline.

<p><b>A</b>. Fluorescence microscopy. Brightfield and fluorescent images of the same cell populations are shown. <b>B</b>. Flow cytometry. In each cell line, with or without added doxycycline, 50,000 cells were analyzed by flow cytometry and plotted as a histogram. Although not shown, unmodified T-REx cells—in the presence or absence of doxycycline—give a distribution that is indistinguishable from uninduced GFP(CAG)₈₉ cells.</p

Relationship between GFP fluorescence intensity and CAG repeat tract length.

<p><b>A</b>. Isolation of cells from different parts of the distribution of fluorescence intensity. Induced GFP(CAG)₈₉ cells were sorted according to the indicated gates, single cells were grown into colonies, and their CAG tracts were amplified by PCR and sequenced. <b>B</b>. Distribution of tract lengths in cells sorted by fluorescence intensity. <b>C</b>. Fluorescence intensity of cells with different CAG tract lengths.</p

Measurements of retinal degeneration.

<p>(A) Counts of nuclei per column of nuclei in the outer nuclear layers (ONL) of retinas from mice with different numbers of wild type rhodopsin genes. Genotypes are abbreviated on the curves: 0 Rho is mRho^−/− (filled squares), 1 Rho is mRho^+/− (open circles), 2 Rho is mRho^+/+ (filled circles), 3 Rho is mRho^+/+NHRE^+/0 (open triangles), and 4 Rho is mRho^+/+NHRE^+/+ (open squares). (B) Counts of nuclei per column of nuclei in the ONL of retinas from mice with the P23H-rhodpsin transgene. Genotypes are abbreviated on the curves: 1 Rho+P23H is mRho^+/−P23H^+/0 (open circles), 2 Rho+P23H is mRho^+/+P23H^+/0 (filled circles), and 3 Rho+P23H is mRho^+/+NHRE^+/0P23H (open triangles). We counted 60–100 columns of nuclei for multiple areas within each retina (3 eyes from 3 individual mice per genotype per timepoint) and averaged them for each time point. Error bars indicate standard error of the mean. Curves were fit to an exponential decay curve, allowing for a plateau value. Exponentials are from non-linear curve fitting using the Marquardt-Levenberg method in Origin, with weighting by 1/variance. The 3 Rho curve-fitting also included data from 3 mice at 8.5 months, which had 6.7 nuclei, and the 4 Rho curve-fitting included data from 1 mouse at 10 months, which had 1.5 nuclei (not shown).</p