FIGURE 5.

Suppression of sirtuin-3 expression replicates the effects of ethanol on Kupffer cell activation. A, Kupffer cells were plated in 6-well plates at 500,000 cells/well. Following a 16-h incubation, the cells were transfected with 50 nm non-targeting siRNA or 25 nm siRNA targeting sirtuin-3 in tandem with 25 nm non-targeting siRNA. After a 24-h incubation, the cells were treated with 100 ng/ml LPS. Alternatively, Kupffer cells were isolated from ethanol-fed mice. Following a 16-h incubation, the cells were transfected with 50 nm non-targeting siRNA. After a 24-h incubation, the cells were left untreated or treated with 100 ng/ml LPS. At the time points indicated, the cells were harvested, and mitochondria were isolated. Mitochondrial lysates were prepared, and sirtuin-3 activity was determined as described under “Experimental Procedures.” Values are the mean of three independent experiments with the error bars indicating S.D. B, 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 25 nm siRNA targeting sirtuin-3 in tandem with 25 nm non-targeting siRNA. After 24 h, 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 exposure. At the time points indicated, aliquots of medium were taken, and the level of IL-1β was determined by ELISA as described under “Experimental Procedures.” 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, the cells were transfected with 50 nm non-targeting siRNA or 25 nm siRNA targeting sirtuin-3 in tandem with 25 nm non-targeting siRNA. After 24 h, 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 exposure. At the time points indicated, the cells were harvested, and caspase-1 activity was determined fluorescently in whole cell lysates. Values are the means 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 25 nm siRNA targeting sirtuin-3 in tandem with 25 nm non-targeting siRNA. After 24 h, 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 exposure. 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 means of three independent experiments with the error bars indicating S.D. E, Kupffer cells were plated in 6-well plates at 500,000 cells/well. Following a 16-h incubation, 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, the cells were pretreated with 20 μm N-HXK II for 30 min prior to LPS treatment. At 2 h, the cells were harvested. Mitochondrial (Mito.) and cytosolic (Cyt.) fractions were prepared and utilized for Western blotting. Densitometry values are indicated below their respective bands and are the mean of three independent experiments ±S.D. AFU, arbitrary fluorescence units; VDAC-1, voltage-dependent anion channel 1.