Fig 2: Ku80 vacates complex heavy-ion-induced DSBs independent of RNF138. U2OS cells proficient (wt) or deficient for RNF138 (RNF138 KO) were irradiated in a low angle (5 × 106 p./cm2) with low-energy carbon (11.4 MeV/nucleon) or high-energy iron ions and fixed 15 min or 1 h after irradiation. Ku80 and 53BP1 (DSB marker) were immunofluorescence stained. Ku80-positive, irradiated (53BP1-positive) cells were quantified. Per sample, at least 100 irradiated cells were analyzed and 6–10 fields of view were enumerated in order to average the number of Ku80-positive cells. Within the irradiated area, all cells were hit by ions. n = 1, error = SEM (a) Fraction of Ku80-positive cells 15 min or 1 h after irradiation in dependence of RNF138. (b) Exemplary images of iron-ion-irradiated wt cells 15 min and 1 h after irradiation and immunofluorescence staining of Ku80 and 53BP1. An enlarged version of the areas highlighted by dashed boxes is shown to the right.

Fig 3: LINP1 associates with NHEJ pathway proteins Ku80 and DNA-PKcs in Hela S3 cells a. Immunoprecipitation efficiency of Ku80 and DNA-PKcs specific antibodies detected by western blots. b and c. Results from RNA-IP. QRT-PCR assays showing the relative level of LINP1 in the complex immunoprecipitated by Ku80- and DNA-PKcs-specific antibodies. d. QRT-PCR assays showing the enrichment of LINP1 with specific biotinylated oligos. e. Western blots showing the Ku80 and DNA-PKcs protein levels in the complexes pulled down by LINP1.

Fig 4: Protein-pulldown experiments point out that the E3 ubiquitin ligases RNF8 and RNF138 are required for radiation-related ubiquitination in G1 cells. (a) HeLa.S-Fucci cells proficient (wt) or deficient for RNF138 (RNF138 KO) and/or RNF8 (RNF8 KD) were irradiated either with 30 Gy X-rays or 30 Gy high-energy iron ions. 1 h after irradiation G1 cells were isolated via flow cytometry, their proteins extracted, and ubiquitinated proteins pulled down and visualized with TUBE2. PD: pull down; IB: immunoblot. (b) For quantification of ubiquitinated proteins, western-blot detection of Ku80 in the input-protein extracts (whole-cell extracts that were used for PD, see (a) was used for normalization to equal loading. For all conditions, the extracts for the pull-down reactions were performed from 106 cells each. Differences in the protein concentration of these extracts are based on the success of cell lysis and reflected in the Ku80 signal. n = 1.

Fig 5: DNA damage in newly born tetraploid S-phase cells is associated with HR and RS-associated markers.(a and c) Diploid and tetraploid RPE-1 S-phase cells labeled with XRCC1 (a, in green) or KU80 antibodies (c, in green). DNA in blue. (b and d) Number of XRCC1 (b) or KU80 (d) foci in diploid and tetraploid RPE-1 cells synchronized in G1 using 1µM palbociclib or released in S-phase. Mean ± SEM, >100 interphase cells were analyzed from at least three independent experiments. (e) Images showing ?H2AX (red) and RAD51 (yellow) foci colocalization (white arrows) in diploid and tetraploid RPE-1 cells. DNA in blue. The white squares correspond to higher magnification regions. (f) RAD51 foci number in diploid and tetraploid RPE-1 cells arrested in G1 using 1 µM palbociclib or released in S-phase. Mean ± SEM, >100 interphase cells analyzed from at least three independent experiments. (g) Percentage of colocalizing ?H2AX and RAD51 signals in diploid and tetraploid RPE-1 cells arrested in G1 using 1 µM palbociclib or released in S-phase. Mean ± SEM, >50 interphase cells were analyzed from at least three independent experiments. (h) Images showing the colocalization (white arrows) of ?H2AX (red) and FANCD2 (yellow). DNA in blue. (i) FANCD2 foci number in diploid and tetraploid RPE-1 cells. Mean ± SEM, >80 interphase cells were analyzed from at least three independent experiments. (j) Graph representing the percentage of ?H2AX signal colocalizing with FANCD2 foci in diploid and tetraploid RPE-1 interphase cells. Mean ± SEM, >50 interphase cells were analyzed from at least three independent experiments. (k) Graph showing RPA number foci in diploid and tetraploid RPE-1 cells arrested in G1 using 1 µM palbociclib or released in S-phase. Mean ± SEM, >100 interphase cells were analyzed from at least three independent experiments. The dotted lines indicate the nuclear area. ANOVA test (two sided) (b, d, f, g, i, j and k). Source data