Fig. 6: Excision pathways that work in parallel with TDP1 in removing TOP1cc are not required for excess DSB generation with TDP1 loss.

a HEK293A-WT, TDP1-KO, XPF-KO, and XPF/TDP1-DKO cells were treated with 10 µM CPT for 1 h. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. Experiments were repeated at least three times, and similar results were obtained. b–e Control siRNA or siRNA against SLX4 (b), MRE11 (c), CtIP (d), or DNA2 (e) were transfected into WT or TDP1-KO cells. 72 h after siRNA transfection, cells were treated with 10 µM CPT for 1 h. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. Experiments were repeated at least three times, and similar results were obtained. f HEK293A-WT, TDP1-KO, TDP2-KO, and TDP2/TDP1-DKO cells were treated with 10 µM CPT for 1 h. Whole-cell extracts were prepared and subjected to Western blotting with the indicated antibodies. Experiments were repeated at least three times, and similar results were obtained. g A neutral comet assay was performed after treating the indicated cells with 1 µM CPT for 1 h. Olive tail movement was measured by open comet software and plotted as a box plot. The center line indicates the median, the box bounds indicate the first and third quartiles, and the whiskers indicate maximum and minimum. Numbers (No.) of cells examined were indicated. A one-way ANOVA Kruskal–Wallis test was used for statistical analysis. ns, not significant. h Control siRNA or siRNA against SLX4, MRE11, CtIP, or DNA2 were transfected into WT or TDP1-KO cells. 72 h after siRNA transfection, a neutral comet assay was performed after treating cells with 1 µM CPT for 1 h. Olive tail movement was measured by open comet software and plotted as a box plot. The center line indicates the median, the box bounds indicate the first and third quartiles, and the whiskers indicate the maximum and minimum. Numbers (No.) of cells examined were indicated. A one-way ANOVA Kruskal–Wallis test was used for statistical analysis. ns not significant.