Nb500 201

Subconfluent cells plated on coverslips were fixed with 3.7% paraformaldehyde (BDH 29447) or in 100% cold methanol. For paraformaldehyde fixed cells, the cells were permeabilized with 0.1 %TritonX-100 in PBS for 15 min at RT and then incubated with blocking solution (1%BSA/10% goat-serum/0.3 M glycine/0.1% Tween in PBS) for 1 h at RT. The cells were incubated overnight at 4 ${}^{\circ }$ C with the primary antibody as following: $\gamma$ -H2AX (1:700, Abcam, ab2893-Phospho139, Rabbit) or anti-survivin (1:250, NB500-201, Novus Biological, Rabbit). The samples were incubated with secondary antibodies FITC anti-Rabbit (1:250, ab150077, AbCam) for 1 h at RT and then mounted with Pro-long anti-fade reagent (P7481, Life Technologies) with DAPI to stain the nuclei. The images were acquired with a Leica TCS NT confocal microscope.

The sections were incubated with a polyclonal anti-Survivin antibody (NB500-201, Novus Biologicals, Littleton, CO, 1:1000) and an anti-PARP6 antibody (HPA026991, Sigma-Aldrich, 1:500). In addition, to analyze the cell proliferation and correlate it with PARP6 and Survivin expression, we examined Ki-67 expression using an anti-Ki-67 monoclonal antibody (MIB-1, Dako). The sections were incubated with the primary antibodies overnight at 4°C after antigen retrieval by microwave treatment in citrate buffer (pH 6.0). The antibodies were detected with the streptavidin-biotin peroxidase system (Universal LSAB™2 kit, Dako, Kyoto, Japan). The immunohistochemistry grade was defined as - to +++ according to the number of stained cells and the intensity of the reaction in the individual cells. The grades were defined as follows: -, the majority of the cells were not positive; +, 5-20% of the tumor cells showed weak to moderate immunoreactivity; ++, 20-50% of the tumor cells showed moderate immunoreactivity; +++, over 50% of the tumor cells showed intense immunoreactivity. The PARP6- and Survivin-positive cases were graded as ++ or +++, and the negative cases were graded as - or +. A labeling index of the percentage of Ki-67-positive cells was determined by examining at least 1000 tumor cells in five representative areas at ×200 magnification.