Anti 53bp1

Cells were grown on coverslips, treated as indicated and fixed in 4% paraformaldehyde for 15 min at room temperature. The coverslips were washed twice with PBS, permeabilized for 5 min in 0.2% Triton X-100, followed by 20 min blocking in filtered 1% BSA in PBS at room temperature. Primary antibody incubations were performed at room temperature for 2 hours. Secondary antibody incubations were performed at room temperature for 30 min. The coverslips were washed twice with PBS, incubated with DAPI (0.5 μg/mL, ThermoFisher) in PBS for 10 min at room temperature, washed three times with PBS and mounted on slides using Mowiol-based mounting media. Primary antibodies used: anti-53BP1 (mouse, 1:10; Schultz et al., 2000 (link)); anti-H2AX Phospho S139 (rabbit, Cell Signaling, 1:500). Secondary antibodies used: Alexa Fluor 488 (goat anti-mouse, ThermoFisher, 1:500); Alexa Fluor 594 (goat anti-rabbit, ThermoFisher, 1:500). Images were acquired with a Zeiss Imager M2 AX10 microscope as described in the section describing detection of mitotic EdU foci.

RIP and dilncRNA analysis was carried out in NIH2/4 as previously described^{16 (link)}, with minor modifications. Briefly, I-SceI-GR-expressing NIH2/4 cells were transfected with the plasmid encoding for N-protein or with an EV as a control at 24 h before triamcinolone acetonide 0.1 µM (Sigma-Aldrich) administration. IP was performed using 5 μg of anti-N-protein, or 10 μg of anti-53BP1 (Supplementary Table 1), or with normal rabbit IgGs (Cell Signaling) as a mock IP.

A549 cells were preincubated in hypoxic conditions (0.2% O₂) for 20 hours, followed by treatment with evofosfamide for 4 hours under hypoxic conditions, reoxygenation and irradiation with 2 Gy. 20 hours after irradiation, cells were washed twice with PBS, fixed with 4% formaldehyde/PBS for 10 min, and washed with PBS (4 × 5 min). Cells were then permeabilized for 5 min with 0.2% ice cold Triton-X-100/PBS, blocked for at least 20 min with 1% BSA, followed by 1 hour incubation with the rabbit monoclonal anti-H2AX-pSer139 (1:100, Abcam, Cambridge, UK) or the rabbit polyclonal anti-53BP1 (1:200, Cell Signaling, Boston MA, USA) primary antibodies, diluted in 1% BSA/PBS. After washing with 1% BSA/PBS (3 × 15 min), cells were incubated with the appropriate secondary antibody diluted 1:1000 (Alexa-488), washed with 1% BSA/PBS (2 × 10 min) followed by PBS (1 × 10 min) and incubated with DAPI/Methanol (1 μg/ml) for 3 min, before fixation with Dako Fluorescent Mounting Medium (Dako, North America). Images were taken using a Leica DM 5500 microscope at a magnification of 40x and quantified using FociCounter software. At least 50 cells/condition were analyzed.

Anti-Nek1 and VS1 [anti-phospho-VDACl (S193)] antibodies were described [17 (link), 22 (link)]. Anti-p84 mAb 5E10, anti-ATM (2C1), anti-ATR were purchased from Genetex (Irvine, CA). Anti-VDAC1 mAb2 and mAb3 was purchased from Calbiochem (La Jolla, CA). Anti-53BP1, Mre11, NBS, Rad51, PSTAIR, and Actin antibodies were purchased from Cell Signaling (Danvers, MA). Anti-CDT1 antibodies were purchased from Bethyl Laboratories (Montgomery, TX). Anti-tubulin (DM1A) antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Fluorochrome-conjugated secondary antibodies (Alexa-Fluoro 488 for green) were purchased from Molecular Probes, Inc. (Eugene, OR), and horseradish peroxidase-based secondary antibodies from Vector Technologies (Burlingame, CA).

For western blot analysis, the cells were lysed in buffer containing 20 mM Tris HCl (pH 7.5), 1 mM Na₂EDTA, 1 mM EGTA, 150 mM NaCl, 1% (w/v) NP40, 1% sodium deoxycholate, 2.5 sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM NA₃VO₄ and 1 μg/ml leupeptin supplemented with complete mini protease inhibitor (Roche, Mannheim, Germany). Denatured proteins (20–40 μg) were electrophoresed in 9% SDS-PAGE gels, transferred onto a nitrocellulose membrane and probed with the specific antibodies anti-HA (MMS-101R, Covance, Berkeley, CA), anti-53BP1 (#4937, Cell Signaling, Danvers, MA, USA), anti-CtIP (rabbit, courtesy of Dr. R. Baer), and anti αTubulin (#T5168, Sigma Aldrich, Munich, Germany). Immunoreactivity was visualized using an enhanced chemiluminescence detection kit (ECL, Pierce).

Immunofluorescence staining was performed as previously described [53 (link)]. The following primary antibodies were used: anti-53BP1 (1:250, #4937, Cell Signaling Technology), anti-phospho-histone H2A.X Ser139 (0.1 μg/mL, #05-636, Millipore, Burlington, MA), tankyrase (1 μg/mL, sc-8337, Santa Cruz Biotechnology) and V5 (0.24 μg/mL, R960-25, Thermo Fisher Scientific). Alexa Fluor 594 or 488-conjugated anti-mouse or rabbit IgG (4 μg/ml, A-11032, A-11029, A-11037 and A-11034, Life Technologies) were used as secondary antibodies. Images were acquired using an Olympus IX71 microscope with a DP80 digital camera (Olympus, Tokyo, Japan) and analyzed by cellSens software (Olympus).