Fig. 2. Propofol overdose induces lysosomal membrane permeabilization (LMP), the loss of mitochondrial transmembrane potential (MTP), and caspase-dependent cell apoptosis. RAW264.7 cells (1 × 106cells/well in 6-well culture plates) were treated with propofol (25 μg/ml) or vehicle for the indicated time periods. (A ) Acridine orange, (B ) rhodamine 123, and (C ) propidium iodide (PI) staining followed by flow cytometric analysis were used to determine the induction of LMP, the loss of MTP, and cell apoptosis, respectively. A representative histogram obtained from three individual experiments is shown, and the percentages of LMP, MTP loss, and apoptotic cells are means ± SD. ***P < 0.001 compared with vehicle. RAW264.7 cells (2 × 105cells/well in 12-well culture plates) were pretreated with (D ) the MTP stabilizer cyclosporin A (CSA, 1 μM) or a drug target control FK506 (1 μM) or (E ) the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-Me)-fluoro methyl ketone (z-VAD-fmk) (20 μM), the caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone (z-IETD-fmk) (20 μM), or the caspase-3 inhibitor benzyloxycarbonyl-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me)-fluoromethyl ketone (z-DEVD-fmk) (20 μM) for 0.5 h followed by propofol (25 μg/ml) or vehicle treatment for 24 h. PI staining followed by flow cytometric analysis was used to detect cell apoptosis. Dimethyl sulfoxide (DMSO) was used as a negative control. The percentages of apoptotic cells are means ± SD of three experiments. ***P < 0.001 compared with the propofol-treated group.