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. 2011 Apr;25(7):717–729. doi: 10.1101/gad.2016111

Figure 6.

Figure 6.

(A) Inhibition of autophagy in 8988T PDAC cells with CQ leads to an increase in DNA damage. The top panels show the results from cells expressing a GFP-53BP1 fusion construct. Note the increase in foci upon CQ treatment, indicating an increase in DNA DSBs. This was mitigated by concurrent treatment with NAC. The histogram below shows quantitation from a representative assay, with a single asterisk representing statistical significance compared with control and a double asterisk representing statistical significance compared with CQ treatment (P < 0.05 Fisher's exact test). (B) Inhibition of autophagy using two siRNAs to ATG5 (siA and B) increases DNA DSBs, measured by 53BP1 foci (charcoal bars) and expressed as percentage of cells with >10 foci. A single asterisk indicates that the increase in 53BP1 foci is statistically significant compared with control (P < 0.05 Fisher's exact test). The light-gray bars show the effect of NAC on 53BP1 foci in a particular experiment. The double asterisk indicates a statistically significant decrease in foci as compared with the corresponding untreated cells (charcoal bar), demonstrating that NAC inhibits the DNA damage caused by autophagy inhibition. (C) 8988T PDAC cells were treated with CQ and subjected to a neutral comet assay to measure the amount of DSBs. Data are expressed as average tail moment (tail moment is the tail length multiplied by the percentage of DNA in the tail). Note the increase upon treatment with CQ as compared with control (asterisk indicates statistical significance; P < 0.05 by t-test). (D) Clonogenic survival assays were performed on 8988T cells transfected with either a control siRNA (charcoal bar) or an siRNA to ATG5 (light-gray bar), and results are expressed as surviving fraction relative to control. Note the significant reduction in surviving fraction with inhibition of autophagy by RNAi. Identical assays were performed in the presence of 1 mM NAC to inhibit ROS. This led to an increase in surviving fraction in ATG5-suppressed cells, indicating a partial rescue by NAC. (E) 8988T cells were treated with CQ with (broken line) or without (solid line) NAC, and proliferation was measured. There was a robust increase in growth with concomitant NAC treatment.