|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 1. TtT/GF cell apoptosis following long-term treatment with TNF
. TtT/GF cells were incubated with culture medium alone (control) or with medium containing 20 ng/ml TNF (TNF) for increasing periods of time. (A) The phase-contrast micrographs show control TtT/GF cells with a typical morphology of spreading cells with abundant filopodia and lamellipodia. After 48-96 hours in culture, the cells became elongated and lamellipodia became less abundant. Following TNF treatment, the cells progressively lost the membrane extensions, they shrunk and detached. Scale bar: 250 µm. Inset: Differential interference contrast microscopy shows membrane blebbings of detached cells that had been treated with TNF for 96 hours. Scale bar: 25 µm. (B) Representative western blots demonstrating the absence and the presence of active caspase 8 in total-cell lysates from control and TNF-treated cells, respectively. (C) Following treatments, total-cell lysates were obtained and subjected to electrophoresis and western blotting with an antibody that exclusively recognises the active caspase 3 fragment and with anti-actin (loading control). The representative western blots show a 17 kDa band corresponding to the active caspase 3 in cells that had been treated with TNF for 72 hours and 96 hours, but not in control TtT/GF cells. (D) TtT/GF cells were incubated with either control culture medium or culture medium containing TNF (20 ng/ml final concentration) both in the absence or in the presence of the caspase inhibitor Z-VADfmk (5 µM, final concentration) for 96 hours. Following treatments, total cell lysates (T), non-cytoskeleton (N) and cytoskeleton (C) fractions were obtained and subjected to electrophoresis and western blotting with an anti-active caspase 3. Cleaved caspase 3 was recovered in the cytoskeleton fraction and the cleavage was blocked by Z-VAD-fmk. (E) Time-course studies showing the increased chromatin condensation (
P<0.001, 72 hours vs 48 hours), nucleosome release (*P<0.0001, 48 hours vs 24 hours) and cell detachment (**P<0.0002, 48 hours vs 24 hours) in TNF-treated TtT/GF cells. Chromatin condensation in apoptotic cells was evaluated by labelling the cells with Hoechst 33342 and propidium iodide. Nucleosome release was measured by quantifying cytoplasmic histone-associated-DNA fragments (mono and oligonucleosomes). Cell detachment was quantified by counting detached cells with a hemocytometer. Data from three independent experiments are expressed as the ratio of treated to control cells ± s.e.m. (F) TNF-induced cell shrinkage and detachment were blocked by caspase inhibition (scale bar: 50 µm). (G) TNF-induced nucleosome release was blocked by the caspase inhibitor Z-VAD-fmk. (H) After incubation with medium alone (control) or containing TNF for increasing periods of time, total cell lysates were subjected to electrophoresis and western blotting with -actinin and spectrin antibodies. The representative western blot shows -actinin downregulation and spectrin cleavage into two fragments of 120 kDa and 150 kDa of molecular mass, respectively (arrows) in TNF-treated cells.
|
