Fig. 7: MUS81 is required for conversion of accumulated TOP1cc into excess DSBs after CPT treatment.

a HEK293A-WT, TDP1-KO, MUS81-KO, and MUS81/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, c Control siRNA or siRNA against EME1 (b) or EME2 (c) 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. d Flow cytometry analysis of pDNA-PKcs-S2056 and DNA contents (PI staining) in WT, TDP1-KO, MUS81-KO, and MUS81/TDP1-DKO cells either not treated (NT) or treated with 10 µM CPT for 1 h. Gates for pDNA-PKcs-S2056-positive cells are shown. The presented numbers are the percentage of pDNA-PKcs-S2056-positive cells (mean ± SD, n = 3 biologically independent experiments). e Quantification of d. The percentage of pDNA-PKcs-S2056-positive cells from three independent experiments were shown in a bar chart (mean ± SD, n = 3 biologically independent experiments). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis. f A flow cytometry analysis of pDNA-PKcs-S2056 intensity in WT, TDP1-KO, MUS81-KO, and MUS81/TDP1-DKO cells treated with 10 µM CPT for 1 h. g Quantification of f. Mean pDNA-PKcs-S2056 intensity from three independent experiments is shown in a bar chart (mean ± SD, n = 3). Two-tailed unpaired t test with Welch’s correction was used for statistical analysis. h 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 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.