There is no clear evidence as to how mammalian AIF is involved in the process of chromatin degradation, but AIF can physi cally interact with DNA and RNA. Interaction of human AIF and endoG, in analogy selleck screening library to what happens in C. elegans, has not yet been shown, although protein analysis in vitro results suggest its possibility. However, other important proteins have been proposed to interact with AIF, namely cyclophilin A and heat shock 70 kDa protein 1A. Cyclophilin A is involved in transport of AIF into the nucleus and may be also involved in chromatin degradation. HSP70 1 binds AIF in the cytoplasm and blocks its transport into the nucleus. Another protein, DNA topoisomerase II , was found to be involved in chromatin degradation during caspase independent apoptosis and is therefore one of our candidate proteins that may interact with AIF in nucleus.

AMID is a flavoprotein with amino acid sequence similarity to NADH oxidore ductases in all species, as well as to AIF. In contrast to AIF, AMID does not contain a recognizable MLS. AMID was found to associate with the outer mitochondrial membrane or to be freely distributed in the cytoplasm. Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries Apoptosis induced by AMID is regu lated by p53, is caspase independent, and is not affected by overexpression of the Bcl Inhibitors,Modulators,Libraries 2 protein. Artificially induced overexpression of AMID caused chromatin con densation at the nuclear periphery and formation of apoptotic bodies. However, this was not confirmed by another study. Overexpression of AMID also caused a loss of structurally differentiated mitochondria.

However, recent results did not show that AMID acts sim ilarly to AIF and Inhibitors,Modulators,Libraries its localization was also questioned. Therefore, in this work we use modern in silico methods for sequence analysis, predicting the subcellular localiza tion of proteins, Inhibitors,Modulators,Libraries prediction of interactions, molecular modeling and docking and we combined these methods with fluorescence microscopic imaging of endoG, AIF, AMID, and other apoptotic proteins in living or fixed cells to analyze their localization and interactions. Materials and methods Cell culture Human bone osteosarcoma U 2 OS cells were grown in minimal essential medium containing Earles balanced salts, L glutamine, non essential amino acids, 1. 5 g l NaHCO3, 10% fetal bovine serum, 100 U ml of penicillin and 100 g ml of streptomycin at 37 C in a 5% CO2 atmosphere.

Plasmid DNA preparation and transfection The plasmid constructs carrying the genes for endoG EYFP, AIF tHcRed, and AMID tHcRed fluorescent fusion proteins were described previously. Cells were trans fected enzyme inhibitor using FuGENE 6. In cases of transient transfection, the transfected cells were used for experi ments 24 hours after transfection. Stably transfected cell cultures were selected using 600 g ml of geneticine for 14 days.