TIP-1 and Radioresistance of Malignant Gliomas Figure 4. TIP-1 facilitates DNA damage repair after IR treatment. A) c-H2AX foci staining. D54 cells were mock treated or irradiated at 5 Gy, and c-H2AX foci were detected by immunofluorescence staining at 0.5, 4 and 24 hours post the treatment. The representative images show positive cells (pointed with arrow heads) containing more than 5 foci. B) Percentage of c-H2AX foci-positive cells was calculated upon at least 100 cells from each of the triplicate experiments. C) Representative images (6400) of IR-induced DNA damage visualized with neutral comet assay. D) Percentage of cells with comet tails (as one indicator of popularity of DNA strand breaks among cells). E) Statistics of tail moments (as one indicator of DNA damage severity in individual cells). At least 75 comet images (including more than 100 cells) were analyzed for each group. Shown are the mean +/2 standard deviation of at least three independent experiments. Significance was determined by comparing the denoted group to the respective vector control with the same treatment, respectively. * p,0.05, t-test. doi:10.1371/journal.pone.0045402.g004 type (WT) or mutant (MUT), was used to co-transfect D54 cells with WT or MUT of c-myc-tagged TIP-1 (Fig. 7D), respectively. The in vivo protein interaction was studied with co-IP of the protein complex from cell lysates followed by SDS-PAGE fractionation and immunoblot with specific antibodies. The results (Fig. 7D) indicated that the protein interaction between TIP-1 and LZAP is mediated by the PDZ domain within TIP-1 and the C-terminal PDZ-binding motif of LZAP. Mutation at either part destroyed the specific protein interaction. TIP-1 enhances the protein interaction between LZAP and ARF in the irradiated glioma cells LZAP was reported as one ARF-interacting protein that modulates HDM2-mediated p53 protein ubiquitination and activation [12]. Co-immunoprecipitation revealed that an ectopic TIP-1 expression enhanced the physical interaction between LZAP and ARF, while TIP-1 knockdown attenuated the association of the two proteins in the irradiated D54 cells (Fig. 7E). In the untreated cells, only a moderate impact of the presence of TIP-1 protein on the LZAP interaction with ARF was observed. It was also noted that, under the experimental PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45402