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Figure 5 Characterization of Macrophage (M) Cell Death Induced by Cycloheximide and Everolimus

(A) Macrophages and smooth muscle cells (SMC) were treated with cycloheximide (10µg/ml) or everolimus (10 µmol/l) for 0 to 24 h. To characterize the type of cell death induced by both compounds, cleavage of procaspase-3 (procasp-3) and internucleosomal DNA fragmentation (both apoptosis markers) were analyzed using Western blotting (upper panel) and agarose gel electrophoresis (lower panel), respectively. SMC treated with the combination of tumor necrosis factor (TNF)-alpha (30 ng/ml) and cycloheximide (CHX, 20 µg/ml) for 12 h served as a positive control. (B) Assaying degradation of long-lived proteins as a biochemical marker for autophagy was studied by treating cells with everolimus (10 µmol/l) or Earle’s balanced salt solution (EBSS) for 12 h. Versus control: **p < 0.01; ***p < 0.001. (C) Western blot analysis of microtubule-associated protein light chain 3 (LC3) processing was performed in both cell types as an alternative method to detect autophagy. Cells were treated with everolimus (10 µmol/l; 0 to 12 h) or EBSS (8 h). All results are representative of 3 independent experiments.