Inhibitory protein TPP1 telomere protection induces ATM-dependent DNA damage response.docVIP

 PAGE \* MERGEFORMAT 2 Inhibitory protein TPP1 telomere protection induces ATM-dependent DNA damage response Authors: Xiao-Lan Guo, Yuan-hua, Zhou Jing-Guo, TANG Zhong, Liu Ning-Tao, Yang, Ching Yu-feng, Wu Fengxia, Zhu Tao Yin [Abstract] Objective: To construct protective protein TPP1 telomere short hairpin RNA expression vector to explore the expression of suppression after TPP1 telomere DNA damage and cell proliferation. Methods: in vitro telomere protective protein TPP1 shRNA retroviral expression vector shTPP1  01, shTPP1  02, and expression of exogenous TPP1 of TPP1  HA plasmid was co-transfected 293T cells, Western Blot test shTPP1 suppression of exogenous TPP1 protein expression; RT  PCR detection of shTPP1 inhibited mouse embryonic fibroblasts (mouse embryonic fibroblast, MEF) endogenous TPP1 mRNA expression. shTPP1 were then infected with ATM + / + MEF and ATM-/ - MEF cells, respectively, using cell growth curve and the BrdU incorporation assay cell proliferation, immune fluorescence / telomere peptide nucleic acid fluorescence in situ hybridization (IF / PNA  FISH) detection of telomere dysfunction-induced damage foci (TIF) formation. Results: shTPP1  01 and shTPP1  02 can significantly inhibit the exogenous and endogenous TPP1 protein TPP1mRNA The expression, shTPP1  01 and shTPP1  02 acting on the primary culture of MEFs grow slowly after the cells, BrdU-positive cells accounted for 8.0% and 8.7%, significantly lower than the control cells in 29.3% (P