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. 2011 Jun 1;22(11):1943–1954. doi: 10.1091/mbc.E10-11-0923

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© 2011 He et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — PIKE-A regulates DNA damage-mediated apoptosis in glioblastoma cells. (A) Depletion of PIKE-A makes glioblastoma cells vulnerable to UV-triggered apoptosis. Activation of caspase-3 was quantified in glioblastoma cells infected with control and sh-PIKE-A adenovirus. Control and sh-PIKE-A adenovirus were used to infect different glioblastoma cells. Forty-eight hours after infection, the cells were exposed to 10 J/m² of UV. Quantitative apoptotic assay in various cell lines is shown. Immunoblotting analysis of PIKE-A knockdown in LN-Z308 cells (right). (B) PIKE-A overexpression represses UV-triggered apoptosis. PIKE-A infected U87-MG, LN229, and SF188 cells revealed the decreased caspase-3 activity, but LN-Z308 and TP366 cells remained the same compared to control. (C) Akt phosphorylation of PIKE-A is required for its prosurvival activity in LN229 cells. LN229 and LN-Z308 glioblastoma cells were transfected with control and various PIKE constructs, followed by UV stimulation. Apoptosis was analyzed after 24 h. (D) PIKE-A is required for FBS-stimulated Akt activation. Wild-type and PIKE-A −/− MEF cells were treated with 10% FBS at various time points. The cell lysates were analyzed with anti–p-Akt 473, anti-Akt, and anti–p-GSK3 antibodies, respectively. (E) Wild-type but not PIKE-A Ser-472A mutant prevents PIKE-null MEF cells from UV-induced apoptosis. Top, wild-type and PIKE-A–null MEF cells were stimulated with UV. After 24 h, the cells were analyzed by immunoblotting with various indicated antibodies and caspase-3 activity was quantified by Caspase-Glo 3/7 assay. Bottom, PIKE-A–null MEF cells were transfected with Myc-PIKE-A WT or Ser-472A followed by UV stimulation. Akt activity and p53 levels were analyzed (bottom left). Caspase-3 activity was quantified by Caspase-Glo 3/7 assay (bottom right). (F) Knocking down of PIKE-A up-regulates p53 and UNC5B, enhancing apoptosis. TP366 cells were infected with control adenovirus or adenovirus expressing shRNA or wild-type PIKE-A. Over 24 h, the infected cells were exposed with UV. After 24 h, the cell lysates were analyzed with various antibodies as indicated.