Article Title: Aberrant DR5 transport through disruption of lysosomal function suggests a novel mechanism for receptor activation
Figure Lengend Snippet: Chloroquine and bafilomycin A abrogate 5-FU generated apoptosis in HCT116 cells through an effect separated from autophagy inhibition The effect of 5-FU with respect to the processing of caspases −8, −3 and PARP was analyzed by immunoblotting using total protein lysates from cells in which autophagy had been compromised by means of chloroquine (CQ, 20 μM), bafilomycin A (Baf A, 100 nM) or 3-methyladenine (3-MA, 5 mM) treatments. Detection of LC3 was performed to verify drug activities A. Cytotoxicity of 5-FU (768 μM, 20h), either used as a single agent or in combination with 10, 20 or 40 μM chloroquine (CQ), was examined by propidium iodide cell labeling followed by FACS analysis of SubG1 populations B. Along with siControl cells, siAtg5 C. siAtg7 D. siBeclin E. and siSQSTM1 (p62) F. transfected (20 nM) HCT116 wt cells were either left untreated or treated with 768 μM 5-FU for 24 h. Western blot examination of cell lysates with respect to cleaved lamin A (cle lamin A), cleaved PARP (cle PARP), p53 and siRNA efficiency are shown in (C–F). The apoptotic effects of 5-FU (768 μM), either alone or in co-treatments using cathepsin inhibitors E64d (5 μM) or pepstatin A (7 μM), or their combination, were analyzed by SDS-PAGE. Immuno-detection of DR5, p53, cleaved caspase-3 (cle casp-3) and LC3 in SDS-PAGE is outlined. G. Expression of DR5 and p53 in HCT116 wt cells as a result of CQ (10, 20, 40 or 80 μM) or 5-FU (768 μM) treatment, or their combinations was investigated by SDS-PAGE using HCT116 wt protein isolates. Immuno-detection of p62 and cleaved PARP served as markers for autophagy and apoptosis, respectively H. Along with controls, HCT116 wt and p5 3 −/− cells were treated with 5-FU (768 μM) or CQ (10 μM), or their combination. Isolated protein lysates were then separated by SDS-PAGE in order to analyze DR5, cleaved PARP, p53 and p62 using specific antibodies I. Immuno-detection of GAPDH or KU80 was used to control for equal loading of samples in (A and C–I). Processed caspase-8 fragments and the short isoform of DR5 are indicated by asterisks (A and G–I). An additional DR5-related fragment detected in western blot and appearing in CQ treated as well as in E64d and pepstatin A co-treated samples is indicated by an arrow (G–I).
Article Snippet: Repeated washes in cold PBS and RNase A treatment (100 μg/ml, Invitrogen) for 1 h at 37°C were followed by propidium iodide staining (50 μg/ml, Sigma-Aldrich).
Techniques: Generated, Inhibition, Labeling, FACS, Transfection, Western Blot, SDS Page, Expressing, Isolation