Figure 5: The internalizing YKWYYRGAA peptide activates cell death via ribosomal protein L29.
From: Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells
(a) Cell viability is reduced on exposure of YKWYYRGAA-pen, relative to complete media, pen, and YKWYYRGAA peptide (1, 3, 10 and 30 μM). The symbol * indicates significant reduction of cell viability (P<0.001; Student's t-test). The MTT assay was performed in triplicate. Bars represent mean±s.e.m. (b) Internalization of YKWYYRGAA peptide induces mammalian cell death. Fluorescence-activated cell sorting of annexin V-FITC-positive cells (apoptotic cells). KS1767 cells were treated with peptides at 30 μM for 6 h at 37 °C. Bars represent mean±s.e.m., from triplicates. (c) Annexin V-FITC binds to translocated phophatidylserine on the plasma membrane of cells treated with YKWYYRGAA-pen. Control, pen, and YKWYYRGAA-treated cells did not translocate the apoptotic marker. Scale bar, 100 μm. (d) The internalizing YKWYYRGAA peptide activates the caspases in the human cell line KS1767. Immunoblotting shows cleavage of caspase-7 (Casp 7) and -9 (Casp 9). Actin served as a loading control. Arrows indicate the caspase fragments. (e) Histone-associated DNA fragments are detected only in cells treated with YKWYYRGAA-pen peptide. DNA fragmentation was detected with the Cell death detection ELISA plus kit (Roche). Bars represent mean±s.e.m., from triplicates. (f) Beclin-1 is upregulated after YKWYYRGAA-pen peptide treatment. Beclin 1, autophagy related protein (atg) 5, and atg-7 expression were detected by western blot. (g) Actin served as a loading control YKWYYRGAA-pen induced the translocation of the high-mobility group protein B1 (HMGB1) into the extracellular environment. KS1767 cells were treated with different peptides at 30 μM or with media as indicated. (h) Normal mitochondria change to vesicular form, and the matrix swells (red arrows). Chromatin condensation and fragmentation (red asterisk) are observed only in KS1767 cells exposed to YKWYYRGAA-pen. Normal mitochondria (white arrows) and nuclei (white asterisks) are observed in control-untreated, pen-treated, and YKWYYRGAA-treated cells. Scale bar, 500 nm.
