FIGURE 3.

Detachment of hexokinase II from the mitochondria and suppression of cyclophilin-D expression prevent LPS- and ethanol-induced Kupffer cell activation. A, Kupffer cells were plated in 6-well plates at 500,000 cells/well. After 16 h, the cells were transfected with 50 nm non-targeting siRNA or siRNA targeting cyclophilin-D (CyP-D). 24 h later, the cells were then treated with 100 ng/ml LPS. Alternatively, the cells were pretreated for 30 min with 20 μm N-HXK II prior to LPS treatment. After a 1-h exposure to LPS, the cells were harvested, and mitochondrial (Mito.) and cytosolic (Cyt.) fractions were prepared for Western blotting. Densitometry values are indicated below their respective bands, and values are the mean of three independent experiments ±S.D. B, Kupffer cells were plated in 24-well plates at 50,000 cells/well. After 16 h, the cells were transfected with 50 nm non-targeting siRNA or siRNA targeting cyclophilin-D. 24 h later, the cells were treated with 100 ng/ml LPS. Alternatively, the cells were pretreated for 30 min with 20 μm N-HXK II prior to LPS treatment. At the time points indicated, aliquots of medium were taken, and the concentration of lactate was determined colorimetrically. Values are the means of three independent experiments with the error bars indicating S.D. C, Kupffer cells were plated in 24-well plates at 50,000 cells/well. Following a 16-h incubation, where indicated, the cells were transfected with 50 nm non-targeting siRNA (siN.T.) or siRNA targeting cyclophilin-D (siCyP-D). After a further 24-h incubation, the cells were treated with 100 ng/ml LPS. Alternatively, cells were pretreated with 20 μm N-HXK II for 30 min prior to LPS treatment. At the time points indicated, the cells were harvested, and whole cell lysates were prepared. Caspase-1 activity was assessed fluorescently as described under “Experimental Procedures.” Values are the mean of three independent experiments with the error bars indicating S.D. D, Kupffer cells were plated in 24-well plates at 50,000 cells/well. Following a 16-h incubation, the cells were transfected with 50 nm non-targeting siRNA or siRNA targeting cyclophilin-D. After a further 24-h incubation, the cells were treated with 100 ng/ml LPS. Alternatively, the cells were pretreated with 20 μm N-HXK II for 30 min prior to LPS treatment. At the time points indicated, aliquots of medium were taken, and the level of IL-1β was determined by ELISA. Values are the mean of three independent experiments with the error bars indicating S.D. E, Kupffer cells were plated in 24-well plates at 50,000 cells/well. Following a 16-h incubation, where indicated, the cells were transfected with 50 nm non-targeting siRNA or siRNA targeting cyclophilin-D. After a further 24-h incubation, the cells were treated with 100 ng/ml LPS. Alternatively, the cells were pretreated with 20 μm N-HXK II for 30 min prior to LPS treatment. At the time points indicated, aliquots of medium were taken, and the level of TNFα was determined by ELISA as described under “Experimental Procedures.” Values are the mean of three independent experiments with the error bars indicating S.D. F, Kupffer cells were plated in 24-well plates at 50,000 cells/well. Following a 16-h incubation, the cells were transfected with 50 nm non-targeting siRNA or siRNA targeting cyclophilin-D. After a further 24-h incubation, the cells were treated with 100 ng/ml LPS. Alternatively, the cells were pretreated with 20 μm N-HXK II for 30 min prior to LPS treatment. Following treatment, the fluorescent probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) was added, and 5 min later the cells were harvested at the time points indicted. Values are the mean of three independent experiments with the error bars indicating S.D.