Figure 8. Cholesterol effect on ASC oligomerization, capase-1 activation, and IL-1β production in the absence of lipin-2. (A) BMDMs from WT and Lpin2−/− mice were treated with 200 ng/ml LPS for 4 h, 2 mM ATP, or both, as indicated. Some samples were preincubated with 100 µg/ml cholesterol 30 min before ATP treatment, as indicated. Proteins from whole-cell lysates and purified cross-linked ASC oligomers were analyzed by immunoblot using specific antibodies against ASC or β-actin, as a loading control. (B) Control (siRNACtrl.) or lipin-2–silenced (siRNALpin2) RAW264.7 cells were treated with 200 ng/ml LPS for 4 h and then 2 mM ATP for 40 min, as indicated. Some samples were preincubated with 100 µg/ml cholesterol (cholesterol/MCD) 30 min before ATP treatment, as indicated. Intracellular active caspase was analyzed by flow cytometry as specified in Materials and methods. Median fluorescence intensities (MFI) are indicated. (bottom) Percentage of cells with active caspase-1. (C) RAW264.7 cells were treated as in B and presence of IL-1β in cellular supernatants was quantified by specific ELISA in triplicate samples. Data are shown as means ± SD, and experiments shown are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01, by Student’s t test.
Figure 8.

Cholesterol effect on ASC oligomerization, capase-1 activation, and IL-1β production in the absence of lipin-2. (A) BMDMs from WT and Lpin2−/− mice were treated with 200 ng/ml LPS for 4 h, 2 mM ATP, or both, as indicated. Some samples were preincubated with 100 µg/ml cholesterol 30 min before ATP treatment, as indicated. Proteins from whole-cell lysates and purified cross-linked ASC oligomers were analyzed by immunoblot using specific antibodies against ASC or β-actin, as a loading control. (B) Control (siRNACtrl.) or lipin-2–silenced (siRNALpin2) RAW264.7 cells were treated with 200 ng/ml LPS for 4 h and then 2 mM ATP for 40 min, as indicated. Some samples were preincubated with 100 µg/ml cholesterol (cholesterol/MCD) 30 min before ATP treatment, as indicated. Intracellular active caspase was analyzed by flow cytometry as specified in Materials and methods. Median fluorescence intensities (MFI) are indicated. (bottom) Percentage of cells with active caspase-1. (C) RAW264.7 cells were treated as in B and presence of IL-1β in cellular supernatants was quantified by specific ELISA in triplicate samples. Data are shown as means ± SD, and experiments shown are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01, by Student’s t test.

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