Fig 2: Telomere length and overhang are not affected by RAP1 depletion in HeLa cells Telomere signal profile of cells treated with or without BIBR1532 +DOX as shown in Fig 4B. The size in kilobases of the highest peak is indicated.STELA assay at the Xp/Yp telomere of HeLa cells treated with the telomerase inhibitor BIBR1532 and depleted for RAP1 (+DOX). The scatter plots show the distribution of STELA products after subtracting 406 bp of flanking DNA, while error bars represent the mean ± standard deviation.Relative mRNA levels of LIG3 and LIG4 measured by RT–qPCR corresponding to the experiment shown in Fig 4C. Data represent mean ± SD of three independent experiments (***P = 0.0001 for shLIG3 and **P = 0.0061 for shLIG4; two‐tailed Student's t‐test).Protein expression of LIG3 and LIG4 used in Fig 4C. The percentage of protein expression is indicated.Telomere overhang assay in HeLa cells. In‐gel hybridization of the telomere probe in native and denaturing conditions. On the right, quantification of the normalized telomere overhang signal performed by dividing the signal intensity obtained by native gel hybridization to the total signal obtained with denatured gels. Data represent one biological replicate.

Fig 3: RECQL4 promotes stabilization of NHEJ machinery at DSBs. (A, B) NHEJ protein–protein interactions are significantly decreased in the absence of RECQL4. DNA-PKcs (A) and Ku70 (B) were immunoprecipitated from U2OS wild-type (WT) or RECQL4 knockout (RQ4KO) cells that were treated with 10 Gy of IR and allowed to recover for 10 minutes. The samples were resolved via SDS-PAGE and interactions between DNA-PKcs and Ku70 with NHEJ factors (LIG4, XRCC4, XLF and/or Artemis) and RECQL4 were determined via immunoblotting. (C, D) RECQL4 promotes recruitment of XRCC4 and XLF to laser-generated DSBs. Relative fluorescent intensity of GFP-tagged XRCC4 (C) and XLF (D) in WT or RQ4KO cells following micro-irradiation are presented as mean ± standard error of the mean (SEM). For XRCC4, the results were calculated from 7 WT and 12 KO cells and 9 WT and 10 RQ4KO cells for XLF. Student's t-test (two-sided) was performed to assess statistical significance (* P< 0.05, ** P< 0.01 and *** P< 0.001). (E) Recruitment of NHEJ core factors to chromatin after IR is attenuated in RECQL4 knockout (RQ4KO) cells. U2OS wild-type (WT) and RQ4KO cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, cytoplasmic, soluble nuclear, and chromatin fractions were isolated for immunoblotting to examine the recruitment of proteins listed in the figure to the chromatin after irradiation.

Fig 4: Telomere fusion assay by PCR ALocation of the primers and probes used for the telomere fusion PCR: Red arrows indicate positions of subtelomeric PCR primers, while black arrows represent the primers used to generate DNA probes for radioactive hybridization with the Southern blot membranes.B, CRepresentative Southern blot membranes of the telomere fusion PCR assay shown in Fig 2A and B. Membranes were subsequently hybridized with the 16p probe, XpYp probe, and finally with the 21q probe.DDifferent RAP1 constructs used in the telomere fusion assay of senescent MRC‐5 cells. The percentage of protein expression is indicated below the blots.EExpression of RAP1, LIG3, and LIG4 related to Fig 2C. The percentage of protein expression is indicated.

Fig 5: DNA-PKi and/or PARPi cotreatment prevents acquired MAPKi resistance in human melanoma cell lines. A–O, Long-term clonogenic growth of isogenic parental and acquired MAPKi-resistant BRAFV600MUT (A–H) or NRASQ61MUT (I–O) human melanoma cell lines showing acquired resistant colonies to BRAFi + MEKi (PLX4032 at 0.5 μmol/L, AZD6244 at 0.5 μmol/L in A and G; PLX4032 at 0.25 μmol/L, AZD6244 at 0.25 μmol/L in D; PLX4032 at 1 μmol/L, AZD6244 at 1 μmol/L in B, C, E, F, and H) or MEKi (trametinib at 0.005 μmol/L in I and 0.01 μmol/L in J and N; trametinib at 0.1 μmol/L in K, L, M, and O) and their suppression by indicated cotreatments with DNA-PKi (NU7026) and/or PARPi (ABT888) at indicated concentrations. Top, representative cultures; bottom, quantifications over n = 4 fields (mean ± SD). Data representative of 2 to 4 independent repeats. Seeding densities (cells/well in 6-well dishes) and culture durations (days): 5,000; 31 (A), 1,000; 17 (B), 5,000; 18 (C), and 40,000; 40 (D). Seeding densities (cells/well in 6-well dishes) and culture durations (days): 5,000; 14 (E), 5,000; 12 (F), 20,000; 30 (G), and 20,000; 21 (H). Seeding densities (cells/well in 6-well dishes) and culture durations (days): 40,000; 30 (I), 50,000; 31 (J), 5,000; 17 (K), 5,000; 22 (L), 5,000; 16 (M), and 20,000; 16 (N). Seeding density (cells/well in 6-well dishes) and culture duration (days): 5,000; 16 (O). Data representative of 2 to 4 independent repeats. P and Q, Western blot, MTT assay (top right), and clonogenic growth assay (bottom) of M245 cells transduced by lentivirus harboring shVector control or shRNAs of PRKDC (P) or LIG4 (Q). TUBULIN, loading control. Clonogenic growth with vehicle (14 days, 5,000 cells/well in 6-well dishes) or MEKi (29 days, 50,000 cells/well in 6-well dishes) treatments. MEKi, trametinib at 0.01 μmol/L. Representative results of 2 independent experiments; quantification of n = 4 fields (mean ± SD). R and S, As in A and J, respectively, except DNA-PKi and/or PARPi cotreatments were performed in M229 (R) and M245 (S) at a later stage of MAPKi treatment (see time-point schema). Inhibitor concentrations for M229: BRAFi + MEKi (PLX4032 at 0.5 μmol/L, AZD6244 at 0.5 μmol/L), NU7026 (8 μmol/L), ABT888 (4 μmol/L), and NU7026 + ABT888 (4 μmol/L + 4 μmol/L). Inhibitor concentrations for M245: MEKi (trametinib at 0.01 μmol/L), NU7026 (5 μmol/L), ABT888 (1 μmol/L), and NU7026 + ABT888 (5 μmol/L + 1 μmol/L). Left, representative cultures; right, quantifications over n = 4 fields (mean ± SD). Data representative of 2 independent repeats. T and U, As in A and J, respectively, except DNA-PKi and/or PARPi cotreatments were performed in M229 (T) and M245 (U) cells at the same concentrations in R and S for the entire duration (day 1–28 in T; day 1–30 in U), the first half (day 1–14 in T; day 1–15 in U) or the second half (day 14–28 in T; day 15–30 in U) of the total treatment course. Top, representative cultures; bottom, quantifications over n = 4 fields (mean ± SD). Data representative of 2 independent repeats. P values (one-way ANOVA followed by the Tukey multiple comparisons test) comparing indicated cultures versus MAPKi-only cultures (A–O and R–U) or shVector culture (P and Q): *, P < 0.05; **, P < 0.01; ***, P < 0.001.