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Fig. 1. Effect of the uptake mechanism on the cargo bioactivity. (A) HeLa cells were either incubated with Smac-Antp for 30 minutes at 37°C or electroporated with Smac with increasing concentrations of each peptide, harvested with trypsin/EDTA and analysed by flow cytometry. Error bars represent the standard deviation of the mean of three independent experiments (a.u., absorbance units). (B) HeLa cells were either incubated or electroporated with medium containing Smac-Antp or Smac (each 5 µM) and analysed by laser scanning microscopy. The left panels show fluorescein fluorescence, the right panels are transmission images. Scale bars: 20 µm. (C) HeLa cells were either incubated with increasing concentrations of Smac-Antp or with Rev-Antp for 30 minutes at 37°C or electroporated with Smac or Rev peptides. After removal of peptides cells were stimulated with the TNF-R1-specific mutant TNF (100 ng/ml) and CHX (2 µg/ml) for 6 hours at 37°C, then scraped off the surface, washed and lysed. Caspase-3 activity was determined in cell lysates using a fluorogenic caspase-3 substrate and expressed as fold activation by dividing the caspase-3 activity in treated cells by the activity in untreated cells (no peptide, no TNF/CHX). Error bars represent the standard deviation of the mean of three independent experiments. (D) Cells were either incubated or electroporated with medium containing peptides. After washing, cells were treated, in triplicate, for 24 hours at 37°C with TNF-R1-specific mutant TNF (100 ng/ml) in the presence of CHX (2.0 µg/ml). Finally, crystal violet staining cell was used as a measure of cell viability.
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