γh2ax ser139

Lungs were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For pathological examination sections were stained with hematoxylin and eosin, according to standard procedures. Antibodies used for immunohistochemistry in lung tumor sections included those raised against: γH2AX Ser 139 (Millipore), Ki67 (Master diagnostica), C3A (Cell Signaling Technology), β-GAL (3A9A; CNIO Monoclonal Antibodies Core Unit, AM(3A9A)) and GFP (Cell Signaling). For β-GAL and GFP double staining, the immunohistochemical reaction was developed using 3,30-diaminobenzidine tetrahydrochloride (DAB) (Chromomap DAB, Ventana, Roche) and purple chromogen (Discovery Purple Kit, Ventana, Roche), respectively. Nuclei were counterstained with Harrys’s hematoxylin. Pictures were taken using Olympus AX70 microscope.

Immunoblotting was carried out as previously described [24 (link)]. For detection of phosphorylated proteins, cell lysates were collected in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with phosphatase inhibitors: 1 mM sodium orthovanadate and 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), as previously described [54 (link)]. Antibodies used include ERK, AKT, MLC2, pERK1/2, pAKT (Ser473), pMLC2 (Ser19) and RhoC (all Cell Signaling, MA, USA), γH2AX (Ser139) from Millipore and RhoA (26C4) from Santa Cruz.

Tissues were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For histological examination sections were stained with hematoxylin and eosin, according to standard procedures as previously described [62 ]. CC3 Cleaved Caspase 3 Asp175 (Cell Signaling Technology 9661), CC10 (Santa Cruz Biotechnology sc-9772), CD4 (Cell Signaling Technology 25229, prosurfactant protein C (millipore AB3786), p21 (291 H/B5, homemade), γH2AX Ser 139 (Millipore 05-636), PPERK Thr202/Tyr204 (Cell Signaling Tehcnology 9378), Ki67 (Cell Signaling 12202), MPO (Dako A0398), F4-80 (ABD Serotec MCA497), p16 (33B, homemade), p19 ARF (sc-32748 Santa Cruz), pRb (ser807/811, #9308, Cell Signaling), pSMAD3 (ser423/425 ab52903, Abcam), pSTAT3 (tyr705, #9145 Cell Signaling), p53 (POE316A, homemade), cyclin D1 (M3635, Dako), c-MYC (ab32072, Abcam), PPARγ (Cell Signaling, #2435), HIF1α (Cell Signaling, #36169), 8-hydroxy-2’-deoxyguanosine (Abcam, ab48508), 4-hydroxy-2-nonenal (Alpha Diagnostic, HNE11-S) antibodies were used for immunohistochemistry in tissue sections. Pictures were taken using Olympus AX70 microscope. The percentage of positive cells was identified by eye and the areas were calculated by ImageJ and Zen 3.1 (Zeiss) softwares.

Immunofluorescence staining of Rad51 (Abcam, ab213, 1:250), γ-H2AX ser139 (Millipore, 05-636, 1:1,000), pRPA (Thr21) (Abcam, ab61065, 1:2,000), BRCA1 (Bethyl laboratories, IHC-00278, 1:500), PCNA (Immuno Concepts, 2037, 1:100), 53BP1 (Millipore, MAB3802, 1:700) were performed as described previously56 (link). GBM cells were grown on GelTrex (Invitrogen)-coated coverslips and treated with shRNA-mediated knock down over a 72 h period. NHA cells were grown to appropriate confluence and treated with 2 mM HU or DMSO vehicle for 2 h. Subsequently, cells were fixed with 4% PFA and immunostained with the indicated primary antibody. Nuclei were counterstained with DAPI (Sigma-Aldrich). Imaging was performed using LSM 700 META/imager.Z1 (plan-apochromat 63 × /1.40 oil DIC M27 objective, Carl Zeiss, Inc.). Confocal images were acquired with equal settings and processed with Zen 2008 software (Carl Zeiss, Inc.). For quantification, 100 non-overlapping images were acquired for each condition using the ScanˆR screening station (Olympus). At least 1,000 cells were scored and processed using ScanˆR Analysis software (Olympus).

Cells were grown on no. 1 glass coverslips and fixed with 4% methanol-free paraformaldehyde (PFA) in PBS. Cells were then permeabilized in PBS plus 0.1% Triton X-100 (PBT) and blocked with normal goat serum plus 0.15 Triton X-100 (NGS-T) followed by incubation with primary antibody in NGS-T overnight at 4°C in a humidity chamber. Coverslips were washed in PBS and incubated with Alexa Fluor secondary antibody (Invitrogen) for 30 min at 37°C. After PBS washes (3×), cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with Gelvatol. The antibodies used were FANCD2 (Novus Biologicals, Inc., catalog no. NB100-182), BRCA1 (Oncogene catalog no. OP92), γH2AX-Ser139 (Millipore catalog no. 05-636), and p-SMC1-Ser957 (Cell Signaling catalog no. 4805). For 4-color immunofluorescence, following secondary washes, cells were incubated with Alexa Fluor 647-γH2AX-pS139 (BD Pharmingen no. 560447) in a light-protected humidity chamber for 1 h at room temperature. Coverslips were washed in PBS, counterstained with DAPI, and mounted in Gelvatol. Cells were imaged using a Zeiss Axioscope and analyzed using ImageJ.