Anti h2ax

BMDM and MEFs were lysed in RIPA buffer and whole cell lysates were generated with LDS sample buffer (Invitrogen) supplemented with dithiothreitol (DTT). For analysis of culture supernatants, protein was precipitated with 7.2% w/v trichloroacetic acid (TCA) (Sigma) followed by two acetone washes. Immunoblotting was carried out as previously described (Helmink et al., 2011 (link)). Primary antibodies used were anti-γ-H2AX clone JBW301 (Millipore) (RRID:AB_309864), anti-H2AX (Millipore) (RRID:AB_2233033), anti-phospho-KAP-1 (Bethyl Laboratories) (RRID:AB_669740), anti-KAP-1 (GeneTex) (RRID:AB_372041), anti-caspase 1 (p20, Casper-1) (Adipogen) (RRID:AB_2490248), anti-DNA-PKcs (Invitrogen), anti-Ku70 (Cell Signaling Technology), anti-Ku80 (Cell Signaling Technology) (RRID:AB_2257526), anti-ATM clone MAT3 (Sigma), anti-Mre11 (Novus) (RRID:AB_10077796), anti-Nbs1 (Abcam) (RRID:AB_777006), anti-Rad50 (Abcam) (RRID:AB_2176935), anti-ATR (Novus) (RRID:AB_10003234), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma). Secondary reagents were horseradish peroxidase–conjugated anti–mouse IgG (Promega) or horseradish peroxidase–conjugated anti- rabbit IgG (Cell Signaling Technology).

Western blotting was performed according to standard laboratory protocols. Antibodies used were: anti-H3K36me3 (Abcam, 9050; 1:1000), anti-H3 (Abcam, 1791; 1:5000), anti-HA (Covance, MMS-101P; 1:2000), anti-H2AX (Millipore 05-636; 1:5000) anti-Tubulin (Abcam, 7291; 1:5000), anti-RCC-1 (Santa Cruz, sc-55559; 1:2000), anti-p21 (Santa Cruz, sc-6246; 1:1000), anti-GAPDH (Santa Cruz, sc-365062; 1:1000), anti-V5-tag (Cell Signaling, 13202; 1:2000), anti-Myc (Abcam, 9106; 1:10000). Secondary antibodies used were: goat anti-mouse HRP (Jackson ImmunoResearch, 115-035-03 or Thermo Fisher Scientific, 31430; 1:5000), goat anti-rabbit HRP (Jackson ImmunoResearch, 111-035-003 or Thermo Fisher Scientific 31460; 1:5000). Uncropped scans of all blots are shown in Supplementary Fig. 12.

Trypan blue, Dulbecco's modified Eagle's medium, RPMI, trypsin, and siRNA were purchased from Life Technologies. Annexin V and DAPI were purchased from Roche Applied Science. Dabrafenib and PLX4032 were purchased from Euromedex. Antibodies against HSP90, E2F1, E2F4, Rb, and phospho-Rb were from Santa Cruz Biotechnology (TEBU, Le Perray en Yvelines, France). Antibodies against PARP, pro-caspase-3, p38, phospho-p38, MDM2, Chk2, p-Chk2, p21, and p27 were from BD Bioscience (Pont de Claix, France) and anti-H2AX was from Merck Millipore (Fontenay-sous-Bois, France).
The E2F1 siRNA number 1 corresponds to a Smart pool (Dharmacon ref: L-003259-00-0005) and the siRNA number 2 was purchassed to Ambion (Ref: 4390824).

The tumour tissues were sliced into frozen sections of 8-μm thickness at −20 °C and were dried in air. The sectioned tissue samples were rehydrated with PBS for 10 min and fixed with 4% (w/v) cold paraformaldehyde for 10 min. The sections were washed with PBS and permeabilised with 0.5% (v/v) Triton^TM X-100 in PBS for 10 min. The sections were blocked in 5% normal goat serum for 30 min at room temperature. The sections were incubated with the following primary antibodies: anti-CD31 (1:500) (eBioscience, CA, USA), anti-NG2 (1:400) (Merck Millipore, MA, USA), anti-ZO-1 (1:400) (Thermo Fisher Scientific, MA, USA), anti-CC3 (1:400) (Cell Signalling Technologies, MA, USA) and anti- H2AX (1:1000) (Merck Millipore, MA, USA) overnight at 4 °C. The sections were washed with 0.1% Tween 20 in PBS and incubated for 1 h at room temperature with the appropriate fluorophore secondary antibody (AlexaFluor 488 or Cy3, goat anti-rat, or goat anti-rabbit IgG; Biolegend, CA, USA). The sections were washed with 0.1% Tween 20 in PBS, dehydrated with ethanol and dried in air; they were then mounted with the Vectashield mounting medium (Vector Laboratories, CA, USA) containing 4′,6-diamidino-2-phenylindole stain (1:5000) and a cover slip.