53bp1 antibody

Isoalantolactone (IATL) was obtained from Chengdu Herbpurify (Chengdu, China). JNK inhibitor SP600125 was purchased from TargetMol (Boston, USA). Doxorubicin (DOX) and N-Acetyl-L-cysteine (NAC) were purchased from Aladdin Industrial Corporation (Shanghai, China). Antibodies of p-JNK, JNK and cleaved-caspase-3 were purchased from Cell Signaling Technology (Danvers, USA). The 53BP1 antibody was provided from Novus Biologicals (Littleton, CO, USA).

The following primary antibodies were used: α-synuclein monoclonal antibody (BD Biosciences), γH2AX, phospho-ATM, ATM, p53, H3, poly(ADP-ribose) polymerase (PARP), ERCC1, XRCC1, MRE11, Rad51, Ku80, DDB2, p16, p73, and H3K9me3 antibodies (Abcam, Cambridge, UK); H2AX, phospho-p53, p21, and Ku70 antibodies (Cell Signaling Technology, Danvers, MA, USA); α-tubulin and β-actin antibodies (Sigma–Aldrich Corp., St. Louis, MO, USA); and 53BP1 antibody (Novus Biologicals, Centennial, CO, USA).
Retinoic acid, poly-L-lysine, and glutathione were obtained from Sigma–Aldrich. NE-PER™ Nuclear and Cytoplasmic Extraction Reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). X-gal was obtained from Duchefa Biochemie (Haarlem, Netherlands).

Cells were seeded onto sterile 16 mm² coverslips placed in six well plates at a density of 1 x 10⁵ cells per well. Cells were left to attach for 4–6 hours before treatment. After incubation with GNPs cells were irradiated with 2 Gy and fixed 1 hour or 24 hours post irradiation with a 50% acetone/50% methanol solution for 10 minutes. Cells were then permeabilised with 0.5% Triton X-100 and PBS solution for 10 minutes before being incubated with a blocking buffer of 0.2% milk, 5% Horse serum, 0.1% Triton X-100 in PBS for 1 hour at room temperature. Coverslips were then incubated with 53BP1 antibody (Novus Biologicals, Colorado, USA) at a dilution of 1:1000 in blocking buffer for 1 hour at room temperature. They were then rinsed three times with washing buffer, 0.1% Triton X-100 in PBS before being incubated with Alexa Fluor 488 Goat anti Rabbit secondary antibody (Invitrogen Molecular Probes, Oregon, USA) at a dilution of 1:1000 in blocking buffer for one hour at room temperature. Coverslips were rinsed three times in washing buffer and then mounted onto glass microscope slides with 5 μl of Vectashield mounting media (Vector Labs Ltd, UK) and sealed with nail varnish. Foci were viewed and counted manually on a Zeiss Axiovert 200 M fluorescent microscope.

IF-FISH was performed as previously described (Sfeir and De Lange, Science 2012). Briefly, cells grown at low density on glass coverslips, prefixed in PBS-1% PFA for 2 minutes, permeabilized in PBS-0.1% Triton X-100 for 15 minutes and then fixed in PBS-4%PFA for 10 minutes. Cells were then saturated in PBS-0.1% Triton X-100-3% fat free milk for 30 minutes, incubated for 2 h with the 53BP1 antibody (Novus Biologicals) and washed three times in the saturation solution, followed by incubation for 1 hour with an anti-rabbit Alexa 594-conjugated IgG antibody (Invitrogen). Cells were then post-fixed for 10 minutes in PBS-2%PFA, dehydrated through an ethanol series and air dried. 0,3ng/μL in 70% formamide 10mM- Tris pH 7.2-1% blocking reagent (Roche) (wash 1 solution) FISH TelC-FITC telomeric probe (PNA Bio) was hybridized at 90°C for 15minutes and 2 hours at 65°C, washed twice in wash 1 solution, washed twice in a 50mM Tris pH 7,2,-150mM NaCl-0,05% Tween 20 solution and nuclear staining was performed with with Hoechst 33342 (Invitrogen). Cells were mounted onto slides with citifluor (Citifluor Ltd.) and imaged using an LSM Exciter confocal microscope (Zeiss) with a 63X plan Apo oil-immersion objective. Images analyses and post-treatment was made using ImageJ software (NIH).

Auranofin (AF), AZD8055 and 3-Methyladenine (3-MA) were purchased from Targetmol (Boston, USA). JNK inhibitor SP600125 was obtained from Selleck Chemicals (Houston, USA). Rapamycin (Rap), everolimus (Eve) and N-acetyl-L-cysteine (NAC) were purchased from Aladdin Industrial Corporation (Shanghai, China). Antibodies of p-JNK, JNK, ATF4, p-eIF2α and eIF2α were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against TrxR1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Antibody of CHOP/DDIT3 and Nrf2 were purchased from Abcam (Cambridge, USA). Antibody of LC3 was purchased from Proteintech (Rosemont, USA). The 53BP1 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Pam3CSK4 and poly(dA:dT) were purchased from Invivogen. DAPI, Antibodies against β‐Actin and β‐Tubulin were purchased from Sigma‐Aldrich. Caspase‐1 p20 antibody (Clone 4B4.2.1) was obtained from Genentech, San Francisco USA. IL‐1β antibody was from R&D Systems. Antibodies against H2A, H2A.X, γ‐H2A.X, P53, and p‐P53(S15) were from Cell Signaling Technology. 53BP1 antibody was obtained from Novus biologicals. Anti‐HA, anti‐GFP, and anti‐BRCA1 antibodies were purchased from Santa Cruz Biotechnology. Pyhin1 and Ifi205b(Mnda) antibody were purchased from Mybiosource. mCherry, Alexa488‐Anti‐Sca‐1 was from Invitrogen and PE/Cy7‐Anti‐cKit, V450‐Anti‐Ly6C, FITC‐Anti‐GR1 were from BD Pharmingen.