Anti par

The following antibodies were used: mouse monoclonal anti-PARP-1, mouse monoclonal anti-BrdU (#347580), mouse monoclonal anti-p21 (#556431), mouse monoclonal anti-p27 (#610241), rabbit polyclonal anti-PAR (#551813,; BD Bioscience), mouse monoclonal anti-α-tubulin (#T5168, Sigma-Aldrich), mouse monoclonal antibodies for Myc (#G019), HA (#G036), Flag (#G191), and GFP (#G096, Abm), rabbit polyclonal anti-MAP2 (#MAB3418), anti-Olig2 (#ab9610), anti-SOX2 (#ab5603, Millipore), anti-GFAP (#ab7260), anti-Ki67 (#ab15580), anti-Tbr2 (#ab183991) and mouse monoclonal anti-Nestin (#ab22035, abcam), goat polyclonal anti-DCX (#sc-8066), anti-NeuroD (#sc-1084), rabbit polyclonal anti-ERK (#sc-93), mouse monoclonal anti-pERK (#sc-7383), anti-LaminB1 (#sc-56145, Santa Cruz), rabbit polyclonal anti-Akt (#4060), rabbit polyclonal anti-pAkt (#4061), rabbit polyclonal anti-PI3K (#4228), anti-pPI3K (#4257), rabbit polyclonal anti-FOXO1 (#2880), rabbit polyclonal anti-pFOXO1 antibody (#9461, Cell Signaling).

After resection of the tumors, they were fixed in neutralized 10% formalin solution and embedded in paraffin blocks using standard procedures. Paraffin sections were stained with hematoxylin-eosin (HE), and histopathological analysis was performed under a light microscopic observation. Tissue sections mounted on slides were also subjected to immunostaining after deparaffinization and rehydration, and antigen retrieval, according to the manufacturer’s protocol. Antibodies used in this study were anti-beta-III-tubulin (Abcam, ab264113, Cambridge, UK), anti-AFP (α-fetoprotein, Pierce, PA5-21004, Shreveport, LA, USA), anti-TRA-1-60 (Santa Cruz, sc-21705, Dallas, TX, USA), Brachyury (Santa Cruz, sc-374321, Dallas, TX, USA) and anti-PAR (BD Pharmingen, Franklin Lakes, NJ, USA). After several washes with PBS, bound antibodies were visualized using 3, 3′-diaminobenzidine according to the manufacturer’s protocol. The sections were counterstained with hematoxylin and mounted.

Protein lysates were prepared via whole cell lysis in ice-cold lysis buffer (150mM NaCl, 10mM Tris-Cl pH 7.4, 5mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (Leupeptin, Pepstatin and PMSF, Sigma Aldridge). Immunoblots were probed with anti-RAD51 (Santa Cruz Biotech), anti-PARP (Millipore), anti-PAR (BD Biosceinces), DNA-PK;P2609 (Cell Signaling) and anti-53BP1 (Bethyl), and with anti-Tubulin (Sigma) and anti-ß-Actin, (Sigma) as controls. Membranes were developed using fluorescent labeled secondary antibodies and visualized using the Odyssey system. Protein expression levels were determined by optical density versus actin loading controls using Image J software (NIH).