Effect of FHC on the occurrence of cell death and on mitochondrial swelling. (A) Jurkat cells (106/mL) were incubated with concentrations of FHC ranging from 12.5 to 100 μM for 20 hours at 37°C. Necrosis and apoptosis were assessed as described in “Materials and Methods” and in Figure 1B (top panel). PARP cleavage was determined by Western blot analysis (bottom panel). (B) Jurkat cells (106/mL) were incubated with FHC (25 μM) for 20 hours at 37°C in the absence or presence of zVAD (50 μM) or CsA (1 μM). Values are means ± SD of at least 4 experiments. Necrosis and apoptosis were assessed as described in panel A. (C) Western blot analysis of cytochrome c release. Jurkat cells were incubated in the absence or in the presence of 50 μM FHC for 20 hours at 37°C. Cell lysates were centrifuged, and the supernatants were analyzed for their cytochrome c content as detailed in “Materials and Methods.” β-actin monoclonal antibody (Clone AC-15) was used as a loading control. (D) Mitochondrial swelling was assayed as reported in Figure 2C. FHC (1 μM) was incubated 1 minute before the addition of 30 μM Ca2+. Mitochondrial swelling was attributed to PTP opening by means of CsA inhibition.
