Fig. 5. Propofol overdose induces glycogen synthase kinase (GSK)-3β-dependent Mcl-1 and glycogen synthase destabilization, lysosomal membrane permeabilization (LMP), mitochondrial transmembrane potential (MTP) loss, and cell apoptosis. (A ) 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. Western blot analysis was used to determine the expression of phospho-GSK-3β (Ser9), GSK-3β, glycogen synthase (GS), and poly (adenosine diphosphate-ribose) polymerase (PARP). (B ) Expression of GSK-3β was silenced in RAW264.7 cells (1 × 10⁶cells/well in 6-well culture plates) using lentiviral-based short hairpin RNA (shRNA) (GSK-3β shRNA; shGSK-3β) constructs and a negative control construct (luciferase shRNA; shLuc). shLuc- or shGSK-3β-transfected cells were treated with propofol (25 μg/ml) or vehicle for 24 h. Western blot analysis was used to determine the expression of Mcl-1, GSK-3α/β, GS, and PARP. β-actin was the internal control. The ratios of these proteins to β-actin are shown compared with the normalized vehicle group. Data are representative of three individual experiments. Meanwhile, RAW264.7 cells (2 × 10⁵cells/well in 12-well culture plates) were pretreated with shGSK-3β (C ), the GSK-3β inhibitor SB415286 (25 μM) for 0.5 h (D ), or the proteasome inhibitor MG132 (0.1 μM) for 0.5 h (E ) followed by propofol (25 μg/ml) or vehicle treatment for 24 h. Acridine orange, rhodamine 123, and propidium iodide staining followed by flow cytometric analysis were used to determine the induction of LMP, the loss of MTP, and cell apoptosis, respectively. Dimethyl sulfoxide (DMSO) was used as a negative control. The percentages of LMP, MTP loss, and apoptotic cells are means ± SD of three individual experiments. ***P < 0.001 compared with the control or propofol-treated group. (F ) RAW264.7 and HepG2 cells (2 × 10⁵cells/well in 12-well culture plates) were pretreated with shGSK-3β and primary human neutrophils (2 × 10⁵cells/well in 12-well culture plates) were pretreated with the GSK-3β inhibitor BIO (10 μM) for 0.5 h followed by propofol (25 μg/ml) or vehicle treatment for 24 h. Annexin V staining followed by flow cytometric analysis was used to determine the induction of cell apoptosis. DMSO was used as a negative control. The percentages of apoptotic cells are means ± SD of three individual experiments. **P < 0.01 and ***P < 0.001 compared with the control group.