Subcellular fractionation T47D buy inhibitor cells and H3396 cells were collected in PBS, centrifuged and resuspended in 200 ul of ice cold fractionation buffer and incu bated on ice for 10 minutes. Cell permeabilization was determined by Trypan blue staining. Cells were then centrifuged at 13,000 rpm and 4 C for 2 minutes. The supernatant containing the cytoplasmic fraction was then isolated from the pellet containing the mitochon drial fraction. The pellet was resuspended in 200 ul RIPA buffer containing 150 mM NaCl, 1% NP 40, 0. 5% deo ycholate, 0. 1% SDS, 50 mM Tris HCl, pH8 and incubated for 30 minutes on ice. Samples were centri fuged for 10 minutes at 13,000 rpm and 4 C. Release of mitochondrial proteins to the cytosol was assessed by SDS PAGE gels and Western blotting.

Measurement of mitochondrial membrane potential Mitochondrial membrane potential was measured by using the fluorescent dye DiOC6 according to the manufacturers instructions. Briefly, after treatment with retinoids, cells were incubated with 50 nM of DiOC6 at 37 C during 30 minutes. Cells were then washed with PBS and trypsi nized. Cells were centrifuged, washed twice with PBS, resuspended in PBS containing 2 ug ml of propidium iodide and analyzed by FACS. Western blotting Caspase 3, 8, 9, cleaved PARP, anti SMAC, anti cytochrome c and b actin antibodies were used to probe blots of e tracts prepared using RIPA buffer. cIAP2 antibodies were used to probe blots of e tracts prepared using Triton Lysis buffer. Immune comple es were detected by chemilumines cence. Plasmids A 1.

4 kb fragment corresponding to the 5 flanking region of cIAP2 gene was amplified by PCR from human genomic DNA and cloned in hoI and NcoI of the basic luciferase reporter plasmid pGL3 Luc. A series of 5deletions of this fragment were amplified by PCR using different forward primers containing a hoI site at their 5 end and a common reverse primer containing a NcoI site at its 5 end. PCR amplified DNA fragments were digested with hoI and NcoI restriction enzymes, gel purified and inserted into the respective sites in pGL3 Luc vector. Site directed mutagenesis of the cIAP2 promoter was performed using QuickChange Site Directed Mutagenesis kit following manufacturer instructions. Nucleotide sequences were determined by automatic DNA sequencer. Information about primer sequences is available upon request. pSG5 I BaSR plasmid was constructed by inserting the human cDNA coding for a constitutively activated form of I Ba containing S32A and S36A mutations from the retroviral plasmid pL SN I BaSR into EcoRI sites of pSG5. Transfection and luciferase assays Transfections Cilengitide were performed using FuGENE transfec tion reagent following manufacturer instruc tions.