|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 10. Effect of PKC
inhibition on TNF
+CHX-induced cell shrinkage and detachment. TtT/GF cells, wild-type and MHCIIB–/– embryonic fibroblasts were incubated with culture medium either alone or with medium containing TNF+CHX both in the absence or presence of the specific PKC inhibitor Myr-PKC pseudosubstrate (10 µM, final concentration) for 16 hours. Some cell preparations were double labelled with anti-active caspase 3 to identify apoptotic cells and with Rhodamine-phalloidin to label F-actin. Next, the total numbers of adherent cells, of flat cells positive for active caspase 3 cells, and cells positive for active caspase 3 cells were recorded for each experimental condition. The representative phase-contrast micrographs on the left of the figure show flat and elongated control TtT/GF, wild-type and MHCIIB–/– cells. PKC-inhibited cells remained attached and showed elongated cytoplasmic processes. TNF+CHX treatment induced cell shrinkage and detachment in TtT/GF cells and wild-type fibroblasts, but MHCIIB–/– cells stayed adherent and elongated. PKC inhibition reduced TtT/GF cell and wild-type embryonic fibroblast detachment induced by TNF+CHX. Scale bar, 100 µm. The histograms on the right of the figure show the quantification of these results. PKC inhibition did not affect cell-death rate in any of the cell lines tested; however, it increased the percentage of flat apoptotic TtT/GF cells and wild-type fibroblasts but was without effect on MHCIIB–/– fibroblasts. The values shown are the mean ± s.e.m. of three independent experiments; more than 300 cells per experimental condition were counted. *P<0.03: TNF+CHX+PKC- vs TNF+CHX-treated wild-type fibroblasts; **P<0.005: TNF+CHX+PKC- vs TNF+CHX-treated TtT/GF cells.
|
