TIP-1 and Radioresistance of Malignant Gliomas Figure 5. TIP-1 suppressed p53 activation after irradiation. D54 cells with an ectopic expression (A) or shRNA depletion (B) were mock treated or irradiated at 5 Gy, whole cell lystates were collected at the indicated time points post the treatment for western blot analyses to profile pathway mediators that sense, transduce, or execute cellular response to DNA damage. C) Semi-quantification of p53 upon the band density on blots using a densitometer. Significance was determined by comparing the denoted group to the respective vector control with the same treatment, respectively. *p,0.05, t-test. D) Analyses of p53 ubiquitination. Cells were mock treated or with IR (5 Gy). MG132 was added at 8 hours post the treatment and incubated for 4 more hours before the cell lysates were collected to analyze the p53 ubiquitination. The ubiquitinated p53 protein was immunoprecipitated with antibody against p53 and blotted with ubiquitin-antibody after the proteins were separated with SDS-PAGE. The representative image shows the ubquitinated p53 ladder (pointed with arrows), as well as overall p53, TIP-1 and actin in each cell lysate. doi:10.1371/journal.pone.0045402.g005 conditions, none of TIP-1, LZAP or ARF was significantly affected by X-ray irradiation at the protein level (Supplementary Figs. S1A, B and unpublished data). LZAP mediates the TIP-1-regulated p53 polyubiquitination To determine roles of the protein interaction between TIP-1 and LZAP in the TIP-1-regulated radioresistance of glioma cells, especially the p53 protein polyubiquitination or accumulation after IR, a panel of siRNAs targeting three independent regions of LZAP gene transcripts was tested in the D54 cells (Fig. 8A). One siRNA (LZAP-1) that most efficiently down regulated the LZAP expression was selected for the rest experiments. The LZAP siRNA was introduced to the D54 cells with variable TIP-1 expression levels. The cells were treated with or without IR (5 Gy) before apoptosis (caspase-3 activation) and p53 ubiquitination assays. Western blot analyses indicated that depleting LZAP with siRNA attenuated the effects of TIP-1 expression on the IR- induced D54 cell apoptosis as indicated with caspase-3 cleavage assays (Fig. 8B). Analyses of the overall p53 protein levels and polyubiquitination status (Fig. 8C) indicated that the impact of TIP-1 expression on the p53 protein accumulation and poly- ubiquitination within the irradiated glioma cells were remarkably reduced in the absence of LZAP protein. TIP-1 knockdown sensitizes D54 xenografts to X-ray radiation in vivo A tumor growth study was conducted to test the feasibility of antagonizing TIP-1 for an improved radiotherapy of malignant glioma by using a mouse model bearing subcutaneous human glioma xenografts. Stable transfected D54 cells with a control shRNA or TIP-1-specific shRNA were subcutaneously implanted PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45402