The effect of lipin-2 on caspase-1 activation. (A) WT and Lpin2^−/− BMDMs (left) or silenced human macrophages (right) were stimulated with 200 ng/ml LPS for 4 h or the indicated periods of time, 2 mM ATP for 40 min, or both, as indicated. IL-18 present in cellular supernatants was quantified by specific ELISAs. (B) Cells as in A were stimulated with LPS and Il18 mRNA levels were quantified by RT-qPCR. (C) Analysis by immunoblot of active caspase-1 present in supernatants from WT and Lpin2^−/− BMDMs (top). (bottom) Densitometric quantification of the bands. (D) Analysis of intracellular active caspase by flow cytometry in RAW264.7 cells treated with control siRNA (siRNACtrl.) or siRNA against lipin-2 (siRNALpin2) and stimulated as in A. Median fluorescence intensities (MFI) are indicated (left). (right) Percentage of cells with active caspase-1. (E and F) WT and Lpin2^−/− BMDMs (E) or silenced human macrophages (F) were pretreated with 10 µM YVAD or ZVAD for 30 min and then stimulated as in A. (G) IL-1β (left) and TNF (right) present in cellular supernatants were quantified by specific ELISAs. LDH release from RAW264.7 cells treated as in D. (H) Analysis by flow cytometry of RAW264.7 cells treated as in D and stained with propidium iodide (PI; left). (right) Percentage of cells positive for PI. Data from A, B, and E–G are shown as mean ± SD, and experiments shown are representative of at least three independent experiments made in triplicate. Data from C, D, and H are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. (E) ^###, P < 0.001, versus Lpin2^−/− + LPS/ATP cells by Student’s t test.