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. 2010 Oct 11;20(1):40–50. doi: 10.1093/hmg/ddq430

Figure 3.

Figure 3.

Endogenous oxidative stress contributes to mitochondrial phenotypes in DJ-1-deficient M17 human neuroblastoma cells. (A) ROS were measured in living cells using the fluorescent dye DFFDA. Under basal conditions, DJ-1-deficient cells (red boxes) had higher DFFDA signal than control lines (black boxes). DFFDA signals were lower, and differences between the lines were not seen when the cells were pretreated for 24 h with 100 μm GSH-EE prior to imaging. Two-way ANOVA was used to compare cell lines and treatments as separate factors and then Bonferroni post hoc tests were used to compare cell lines for each treatment; ***P < 0.001; ns, not significant. (B) TMRE and DAPI were used to estimate Δψm and nuclear integrity, respectively, and measured using flow cytometry. Controls (upper panels) had higher TMRE staining than DJ-1-deficient cells (lower panels). Treatment with GSH-EE (right pair of panels) increased the proportion of DJ-1-deficient cells with higher TMRE staining. (C) Cells were treated with GSH-EE and Δψm estimated by imaging of live cells using TMRE. DJ-1 shRNA lines (red) bars had lower TMRE signals compared with control lines (open bars) and this difference was diminished after treatment with GSH-EE. Two-way ANOVA was used to compare cell lines and treatments as separate factors and then Bonferroni post hoc tests were used to compare cell lines for each treatment; ***P < 0.001; ns, not significant. (D) Mitochondrial morphology in controls (upper panels) or DJ-1-deficient cells (lower panels) was imaged after transfection with plasmids encoding mitochondrially targeted YFP. Addition of GSH-EE caused a change in the morphology of mitochondria in DJ-1-deficient cells toward a more elongated, connected phenotype. Scale bar is 2 μm. (E) Quantitation of mitochondrial connectivity using FRAP shows a recovery of signal in the DJ-1-deficient cells (red boxes) after treatment with the glutathione precursor. Statistical significance was calculated using two-way ANOVA with Bonferroni post hoc tests to compare cell lines; ***P < 0.001; ns, not significant (n = 60 cells per condition from duplicate experiments). (F) Counts of LC3-GFP-positive punctatae as a marker of autophagy show that the difference between controls and DJ-1 shRNA lines is diminished after GSH-EE treatment. ***P < 0.001; ns, not significant by χ2-test, n = 3 experiments with >50 cells counted per experiment.