The results of the present study demonstrate that PMA mediated PKC activation strongly increases apicu laren A induced apoptotic cell death and disruption of microtubule networks in HeLa cells. Methods Cell culture Human HeLa cervical cancer cells were cultured in Dulbeccos modified Eagles medium sup plemented with concerning 10% fetal bovine serum and antibiotics. Cells were maintained at 37 C, 5% CO2 and 95% air. Antibodies and chemicals Apicularen A was provided by Dr. Ahn and dissolved in dimethyl sulfoxide. Phorbol 12 myristate 13 acetate, thiazolyl blue tetrazolium bromide, anti tubulin and anti B tubulin antibodies were pur chased from Sigma. Anti PARP and anti actin antibodies were purchased from Santa Cruz Biotechnology. Anti caspase 3 antibody was purchased from R D Systems.

Inhibitors,Modulators,Libraries Z VAD fmk, Ro31 8220 and Go6983 Inhibitors,Modulators,Libraries were purchased Inhibitors,Modulators,Libraries from Calbiochem. All other reagents were molecular biology grade. Cell viability assay Cell viability was assessed by thiazolyl blue tetrazolium assay. Exponentially growing cells were exposed to apicularen A in the presence or absence of PMA for 24 and 48 hours. MTT solution was added to each well and incubated for 2 hours. Cell viability was assessed by measuring the absorbance at 570 nm in an ELISA plate reader. DNA fragmentation assay The cells were lysed using buffer containing 300 mM Tris HCl, 100 mM NaCl, 10 mM EDTA, 200 mM su crose and 0. 5% SDS. Intracellular DNA was extracted with phenol chloroform and chloroform isoamylalcohol. DNA was precipitated and digested in 10 mM Tris HCl, 1 mM EDTA and 40 ug ml RNase A for 1 hour at 37 C.

Then, DNA was resolved by electrophoresis in a 1. 2% agarose gel supplemented with ethidium bromide, and DNA fragmentation was examined by ultraviolet transillumination. Inhibitors,Modulators,Libraries Caspase 3 activity assay Cell extracts were prepared by suspending 2 106 HeLa cells in 100 uL TTE buffer on ice for 30 min, Inhibitors,Modulators,Libraries and then centrifuging at 15,000 g for 10 minutes at 4 C. Ly sates were mixed with 90 ul assay buffer containing 40 uM Ac DEVD AFC. Caspase 3 activ ity was measured at 37 C using a spectrofluorometric plate reader in kinetic mode using excitation and emission wavelengths of 400 nm and 505 nm. Western blotting analysis HeLa cells were lysed in buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% nonidet P 40, 0. 5% deoxycholate, 0. 1% SDS and protease inhibitor cocktail. Cell ly sates were subjected to SDS PAGE and transferred AZD9291 solubility onto nitrocellulose or PVDF membranes. The membranes were first probed with primary antibodies and then with HRP conjugated secondary antibodies, and the proteins were detected using the ECL system. Cell cycle analysis HeLa cells exposed to apicularen A in the presence or absence of PMA were washed with phosphate buffered saline and fixed in 70% ethanol at 20 C over night.