TIP-1 and Radioresistance of Malignant Gliomas Figure 8. LZAP is required for the TIP-1-regulated IR-induced apoptosis and p53 polyubiquitination in D54 glioma cells. A) Western blot analysis of LZAP knockdown in D54 glioma cells with siRNAs. Whole cell lysates were analyzed at 72 hours post the transfection. A validated control siRNA was included. B) Western blot analysis and quantification of caspase-3 cleavage with or without LZAP knockdown. Cells were transfected with LZAP-targeting siRNA or a control siRNA 72 hours prior to irradiation at 5 Gy. Whole cell lysates were analyzed at 24 hours post IR treatment. Significance was determined by comparing the denoted group to the respective vector control. *p,0.05; ** p.0.05 with t-test. C) Western blot analysis of the p53 protein levels and ubiquitination with or without LZAP knockdown. The cells were irradiated at 5 Gy. MG132 was added at 8 hours post IR treatment and incubated for 4 more hours before the cell lysates were collected to analyze the p53 ubiquitination. The representative image shows the ubquitinated p53 ladder (pointed with arrows) as well as overall LZAP, p53, TIP-1 and actin in each cell lysate. doi:10.1371/journal.pone.0045402.g008 Figure 9. TIP-1 depletion sensitized D54 tumor xenografts to IR in vivo. Stable transfected D54 cells with a control or TIP-1 targeting shRNA were implanted subcutaneously in hind limbs of Foxn1-null nude mice. Tumor sizes were measured with caliper, and irradiation (3 Gy for five consecutive days as indicated with arrows) was delivered to tumors when the tumor size reached 3 mm in diameter. n = 9 in each group. doi:10.1371/journal.pone.0045402.g009 PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e45402