Cells transfected with the empty vector (pRluc) (Fig. 9). Altogether these data suggested that 23388095 the C-terminal extension of aA-crystallin was sufficient to promote the anti-apoptotic action of the protein JW 74 biological activity through binding with Bax and preventing its activation and translocation to the mitochondria.a-Crystallin Cytoprotective ActionFigure 5. Stable expression of a-crystallins in 661W cells. (A) Schematic representation of the bicistronic pWPI lentiviral vector allowing for pEF1a-driven simultaneous expression of the transgene (aA- or aB-crystallin) and IRES-mediated GFP fluorescent marker. (B) Western blot analysis of aA- and aB-crystallins expressed in lentivirus-transduced 661W cells. Twenty-five micrograms of total proteins from cell extracts were subjected to 12 SDS-PAGE. Myc-tagged aA- and aB-crystallins were expressed in 661W cells transduced with the recombinant lentiviruses pWPI_aA and pWPI_aB, respectively, while no transgene was detected in cells transduced with the empty lentivirus pWPI or in non transduced cells (2). Membranes were further immunoassayed with anti-GFP as a control of transduction efficiency and with anti-?actin as a control of equal protein loading. (C) Immunofluorescence analysis with anti-myc showing cytoplasmic expression of aA- and aB-crystallins in 661W cells transduced with the corresponding recombinant lentiviruses. Cell nuclei were counterstained with DAPI and GFP staining was shown as a control of transduction efficiency. pEF1a: EF1a promoter; IRES: internal ribosome entry site from encephalomyocarditis virus. doi:10.1371/journal.pone.0055372.gDiscussionIn the current study, we reported the anti-apoptotic action of aA- and aB-crystallins against Bax-triggered apoptosis. Indeed, caspase-induced apoptosis was inhibited in 293T cells overexpressing a-crystallins, reflected by the inhibition of Caspase-3/-activity and the decrease in TUNEL-positive apoptotic cells. By co-immunoprecipitation study, we further showed that aA- and aB-crystallins directly interacted with pro-apoptotic Bax in vivo, suggesting that a-crystallins exert their pro-survival action by sequestering Bax in the cytoplasm to prevent its activation anda-Crystallin Cytoprotective ActionFigure 6. STS-induced apoptosis was prevented in 661W cells in the presence of a-Crystallins. 661W cells transduced with the recombinant lentiviruses overexpressing aA-crystallin 15857111 (pWPI_aA), aB-crystallin (pWPI_aB) or the empty lentivirus (pWPI) were treated with 100 nM STS for 16 h. (A) STS-triggered apoptosis was inhibited in the presence of a-crystallins, as reflected by TUNEL assay using TMR-dUTP. (B) STS-induced caspase activation was decreased in 661W cells overexpressing aA- and aB-crystallins, as measured by colorimetric Caspase-3/-7 assay. (* p,0.005 by t-test for pWPI versus pWPIaA). Data are the mean 6 SE of four independent experiments. doi:10.1371/journal.pone.0055372.gtranslocation to the mitochondria. In support of this, the overexpressed aA- and aB-crystallins were essentially localized in the cytoplasm of the transfected cells. In lens-derived ML-240 custom synthesis epithelial cell line, a-crystallins have been shown to inhibit STS-induced apoptosis through interactions with members of the Bcl-2 family. Through binding to Bax and Bcl-Xs, aA- and aB-crystallins prevented the translocation of the pro-apoptotic proteins from cytosol into mitochondria, repressing the release of cytochrome C and the activation of Caspase-3 upon STS treatment [13]. Pasupuleti et al. demo.Cells transfected with the empty vector (pRluc) (Fig. 9). Altogether these data suggested that 23388095 the C-terminal extension of aA-crystallin was sufficient to promote the anti-apoptotic action of the protein through binding with Bax and preventing its activation and translocation to the mitochondria.a-Crystallin Cytoprotective ActionFigure 5. Stable expression of a-crystallins in 661W cells. (A) Schematic representation of the bicistronic pWPI lentiviral vector allowing for pEF1a-driven simultaneous expression of the transgene (aA- or aB-crystallin) and IRES-mediated GFP fluorescent marker. (B) Western blot analysis of aA- and aB-crystallins expressed in lentivirus-transduced 661W cells. Twenty-five micrograms of total proteins from cell extracts were subjected to 12 SDS-PAGE. Myc-tagged aA- and aB-crystallins were expressed in 661W cells transduced with the recombinant lentiviruses pWPI_aA and pWPI_aB, respectively, while no transgene was detected in cells transduced with the empty lentivirus pWPI or in non transduced cells (2). Membranes were further immunoassayed with anti-GFP as a control of transduction efficiency and with anti-?actin as a control of equal protein loading. (C) Immunofluorescence analysis with anti-myc showing cytoplasmic expression of aA- and aB-crystallins in 661W cells transduced with the corresponding recombinant lentiviruses. Cell nuclei were counterstained with DAPI and GFP staining was shown as a control of transduction efficiency. pEF1a: EF1a promoter; IRES: internal ribosome entry site from encephalomyocarditis virus. doi:10.1371/journal.pone.0055372.gDiscussionIn the current study, we reported the anti-apoptotic action of aA- and aB-crystallins against Bax-triggered apoptosis. Indeed, caspase-induced apoptosis was inhibited in 293T cells overexpressing a-crystallins, reflected by the inhibition of Caspase-3/-activity and the decrease in TUNEL-positive apoptotic cells. By co-immunoprecipitation study, we further showed that aA- and aB-crystallins directly interacted with pro-apoptotic Bax in vivo, suggesting that a-crystallins exert their pro-survival action by sequestering Bax in the cytoplasm to prevent its activation anda-Crystallin Cytoprotective ActionFigure 6. STS-induced apoptosis was prevented in 661W cells in the presence of a-Crystallins. 661W cells transduced with the recombinant lentiviruses overexpressing aA-crystallin 15857111 (pWPI_aA), aB-crystallin (pWPI_aB) or the empty lentivirus (pWPI) were treated with 100 nM STS for 16 h. (A) STS-triggered apoptosis was inhibited in the presence of a-crystallins, as reflected by TUNEL assay using TMR-dUTP. (B) STS-induced caspase activation was decreased in 661W cells overexpressing aA- and aB-crystallins, as measured by colorimetric Caspase-3/-7 assay. (* p,0.005 by t-test for pWPI versus pWPIaA). Data are the mean 6 SE of four independent experiments. doi:10.1371/journal.pone.0055372.gtranslocation to the mitochondria. In support of this, the overexpressed aA- and aB-crystallins were essentially localized in the cytoplasm of the transfected cells. In lens-derived epithelial cell line, a-crystallins have been shown to inhibit STS-induced apoptosis through interactions with members of the Bcl-2 family. Through binding to Bax and Bcl-Xs, aA- and aB-crystallins prevented the translocation of the pro-apoptotic proteins from cytosol into mitochondria, repressing the release of cytochrome C and the activation of Caspase-3 upon STS treatment [13]. Pasupuleti et al. demo.
