en in ” aB crystallin KO RPE exposed to the same concentration of H2O2. Results a-crystallin overexpressing RPE cells are resistant to oxidative stress induced cell death We generated a-crystallin overexpressing stable cell lines and demonstrated that aA crystallin or aB crystallin overexpressing cells were more resistant to H2O2-induced cell death than vector control cells. Overexpression of aA crystallin or aB crystallin resulted in 10% cell death at concentrations of H2O2 that caused 30% cell death in control cells. Further, caspase 3 activation was inhibited in acrystallin overexpressing cells exposed to H2O2. The dose and duration of H2O2 used in these studies were 150 mM and 24 h, respectively, as has been validated in our previous work. Higher thiol levels provide protection from oxidative stress in a-crystallin overexpressing cells We next investigated the link between a-crystallin expression, intracellular thiol levels and enhanced cell survival in oxidative stress. Our data revealed a significant 2-fold increase in cellular GSH levels in a-crystallin overexpressing clones when compared to controls. One of the main mechanisms for elevation of cellular GSH is increased biosynthesis catalyzed by the rate-limiting enzyme glutamate-cysteine ligase . The increase in total GSH levels was associated with significant upregulation of the gene and protein expression of the catalytic unit of GCL but not GCLM, the modifier unit of GCL. Mitochondrial fractions from a-crystallin overexpressing cells had significantly higher GSH levels after treatment with 150 mM H2O2 for 24 h. The Aphrodine chemical information magnitude of increase in GSH level in cytosol, MRP-related GSH transporters in RPE 17622149” cells We then proceeded to characterize the transporter mediating GSH efflux from RPE cells. Several MRPs are known to mediate GSH efflux in mammalian cells. To determine the presence of MRPs in RPE, MRP mRNA levels were analyzed by RT-PCR. RNA isolated from RPE cells was amplified using specific MRP primer sequences. mRNAs encoding for MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, and MRP7 were detected in RPE cells. MRP1 was the most abundant of the MRP family members in RPE. All further experiments were performed with MRP1 because it is the most well 
characterized MRP with respect to efflux of GSH and GSSG. MRP1-Mediated GSH Efflux in RPE Cells Localization of MRP1 in a-Crystallin overexpressing RPE cells In subconfluent ARPE cells, MRP1 is predominantly localized in the plasma membrane and the staining pattern is punctate. In human polarized RPE monolayers, we observed lateral membrane localization of MRP1. Biotinylation of intact cells with subsequent immunoblot analysis revealed surface localization of MRP1 in the membrane fraction. These studies further established that membrane expression of MRP1 was almost three fold higher in aB crystallin overexpressing cells than vector control cells which correlated well with the increased GSH efflux in a-crystallin overexpressing cells. In addition, cellular MRP1 expression showed a.2.5 fold increase in aB crystallin overexpressing cells as compared to vector control cells. Furthermore, consistent with GSH efflux under oxidative stress, we observed a.2 fold increase in MRP1 expression only in vector control cells subjected to oxidative stress. Having established that increased a-crystallin levels increased MRP1 expression, we then investigated whether knocking down of aB crystallin could affect the expression of MRP1. As seen in Fig. 5E, a signific
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