Fig. 2: Trabectedin-induced DNA break formation and toxicity depend on the catalytic activity of XPF but not that of XPG.

a HAP1 WT, ERCC1-KO, and XPF-D687A cells were treated with trabectedin for 2 h and colonies counted after 7 days. Mean ± SEM of 5 (WT, XPF-D687A) and 3 (ERCC1-KO) biological replicates (3 technical replicates per experiment). Provided p-values are derived using ordinary two-way ANOVA with Dunnett’s multiple comparisons test (between WT and ERCC1-KO or XPF-D687A at each concentration). b HAP1 WT, ERCC1-KO, and XPF-D687A cells were arrested in G1 with palbociclib and treated with trabectedin (50 nM, 2 h) and incubated for up to 4 h with or without repair synthesis inhibitors (0.5 mM HU, 5 μM AraC). ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 3 biological replicates (Supplementary Fig. 2c). Provided P-values are derived using two-tailed paired t-test (between WT and ERCC1-KO or XPF-D687A at each recovery time). c XP2YO patient cells were treated with trabectedin for 2 h and colonies were counted after 8 days. Mean ± SEM of 2 biological replicates (3 technical replicates per experiment). d U2OS WT and XPF-KO cells were arrested in G1 with palbociclib and treated with trabectedin (50 nM, 2 h) and allowed to recover for up to 4 h with or without repair synthesis inhibitors (1 mM HU, 10 μM AraC). ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 4 biological replicates. P values were derived using ordinary two-way ANOVA with uncorrected Fisher’s LSD (between WT and XPF-KO at each recovery time). e HAP1 WT, XPG-KO, and XPG-E791A cells were treated with trabectedin for 2 h and colonies counted after 7 days. Mean ± SEM of 4 biological replicates (3 technical replicates per experiment). Provided P-values are derived using ordinary two-way ANOVA with Dunnett’s multiple comparisons test (between WT and XPG-KO or XPG-E791A at each concentration). f HAP1 WT, XPG-KO, and XPG-E791A cells were arrested in G1 with palbociclib (2 µM, 24 h) and treated with trabectedin (50 nM, 2 h). Cells were kept in G1 with or without repair synthesis inhibitors (0.5 mM HU, 5 μM AraC) and incubated for up to 4 h. ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 5 biological replicates (Supplementary Fig. 3d–e). Provided P-values are derived using ordinary two-way ANOVA with Tukey’s multiple comparisons test (between WT and XPG-KO or XPG-E791A at each recovery time). g A simplified schematic of assessing NER incision activity on trabectedin-induced DNA adducts in HAP1 WT, XPF-D687A, ERCC1-KO, XPG-E791A, and XPG-KO cells using alkaline COMET chip assays. Source data are provided as a Source Data file.