In the present study we tested this hypothesis by investigating whether TCTP involves in the development of chemoresistance in mitochondria mediated apoptosis. We specifically examined the role of TCTP in apoptosome inhibition, by studying its structural modification in etoposide treated cancer cells. Methods Reagents and antibodies Antibody detecting anti Na,K selleck chemicals ATPase 1 subunit was pur chased from Upstate. Anti PLC, actin, His, cytochrome c, caspase 9, cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved PARP, Flag, and Apaf 1 antibodies were from Cell Signaling Technology. Anti EGFR, and GFP antibodies were from Santa Cruz. Anti TCTP specific antibody was from LabFrontier. tet iodide was from Molecular Probe. Etoposide, Taxol, Ac LEHD and Ac DEVD were from Calbiochem.

Bovine serum al bumin, dATP, cytochrome c, and carbonyl cyanide m chlorophenylhydrazone were from Sigma. Inhibitors,Modulators,Libraries Anti OxPhos Complex IV antibody was from Invitrogen. Purified WD Repeat protein was from Abnova Corporation. Cell culture and infection HeLa cells that were from American Type Culture Inhibitors,Modulators,Libraries Collection were maintained and cultured in a Dulbeccos modified Eagle medium supple mented with 10% fetal bovine serum, penicillin, and streptomycin. Cells were placed at 37 C in a 5% CO2 atmosphere incubator with humidification. For adenoviral expression, cells were infected with 10 multiplicity of infection of adenoviruses containing N terminal Flag tagged TCTP or C terminal GFP tagged TCTP genes, Inhibitors,Modulators,Libraries or with its corre sponding null virus for 2 h in DMEM without serum at 37 C in 5% CO2, followed by 20 h incubation in DMEM media containing 10% serum.

The cells were then serum starved for 2 h prior to drug treatment. The level of TCTP overexpression was determined by western blot analysis. Etoposide was administered Inhibitors,Modulators,Libraries after infection of adenovirus. Cell death analysis For detection of apoptosis, HeLa cells were seeded onto 12 well plates and treated with etoposide or taxol for an indicated time. To measure the DNA fragmentation by apoptosis, cells were stained with propidium iodide and were assayed under fluorescence activated Inhibitors,Modulators,Libraries cell sorting analysis. Following the treatment with cytotoxic agents, HeLa cells were harvested, and reconstituted in ice cold phosphate buffered saline supplemented with 50 ug ml of PI. Samples were then detected their fluorescence by flow cy tometry and the results were analyzed using WinMDI software. Immunoprecipitation and western blotting Under the presence of dATP and cytochrome c, HeLa S 100 extract till was incubated with recombinant human TCTP for 1 h at 4 C in PBS. The reaction mixtures were subjected to preclearance by adding Protein G agarose and incubated for 3 h at 4 C on a rocking platform, to remove the non specific pro tein binding to agarose.