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. 2015 Sep 25;290(46):27986–28000. doi: 10.1074/jbc.M115.676437

FIGURE 2.

FIGURE 2.

DNA double-strand breaks occur in aggresome-containing cells. A, confocal microscopy showing immunostaining of γ-H2AX. Long term (three month) expression of EGFP-HDQ72 led to the formation of γ-H2AX foci in the nuclei of cells that contain aggresomes. Positive control: 3-month cultures of EGFP-HDQ23 cells treated with 25 μm etoposide for 8 h. Negative control: untreated 3-month cultures of EGFP-HDQ23 cells. B, quantitation of cells with γ-H2AX foci in nuclei (from confocal microscopy data). Half (50.4%) of aggresome-containing EGFP-HDQ72 cells had DSBs, whereas this proportion was much reduced (∼15%) in EGFP-HDQ72 cells without aggresomes and in negative control EGFP-HDQ23 cells. Three independent experiments are represented with ∼200 EGFP-HDQ72 or EGFP-HDQ23 cells assessed per experiment. The chart represents the mean ± S.D. Samples were analyzed by binary logistic regression. ***, p < 0.001; ns, not significant. C, immunoblotting of γ-H2AX in long term (3 months) cell cultures. Left panel, an elevated level of γ-H2AX was present in EGFP-HDQ72 cells (EGFP-HDQ72) compared with the negative controls, uninduced EGFP-HDQ72 cells (UN), and EGFP-HDQ23 cells (EGFP-HDQ23). The positive control was long term-induced EGFP-HDQ23 cells treated with 25 μm etoposide for 8 h. D, γ-H2AX protein level correlates with polyQ expression level. SNAP-HDQ72 expression of long term-induced cells (3 months) was driven from either weak (W), intermediate-strength (I), or strong (S) promoters. UN (−) represents uninduced SNAP-HDQ72 cells (strong promoter) as the negative control. E, quantitation of cells with γ-H2AX foci in the nucleus. Confocal microscopy shows that the proportion of cells containing γ-H2AX foci correlates with polyQ expression levels controlled by weak (W), intermediate-strength (I), or strong (S) promoters. Samples are from cells with long term (3 months) EGFP-HDQ72 expression. Three independent experiments were performed with ∼200 cells assessed per population per experiment. The chart represents the mean ± S.D. Samples were analyzed by binary logistic regression. ***, p < 0.001; ns, not significant. F, γ-H2AX levels increased after expression of EGFP-HDQ72 for 2 weeks. γ-H2AX levels increased during a longer period (5 weeks) of consistent expression of EGFP-HDQ72. UN (−) represents uninduced SNAP-HDQ72 cells (strong promoter) as the negative control. G, γ-H2AX levels increased after expression of EGFP-HDQ72 for 2 weeks and reached peak levels after 4 weeks.