The effect of lipin-2 on IL-1β production by murine and human macrophages. IL-1β presence in supernatants from primary human macrophages (A), BMDMs from WT and Lpin2^−/− mice (B and D), or RAW264.7 cells (C) stimulated with 200 ng/ml LPS for 4 h, 2 mM ATP for 40 min, or both, as indicated, were quantified by specific ELISAs. (A–C) Human macrophages and RAW264.7 cells were silenced as described in Materials and methods, using control (siRNACtrl.) and lipin-2–specific siRNAs (siRNALpin2). (right) Lpin2 mRNA levels at the time of stimulation. (D) BMDMs supernatants were analyzed by immunoblot using specific antibodies against IL-1β. (right) Densitometric quantification of the bands. (E) IL-1β production was analyzed in BMDMs supernatants from WT, Nlrp3^−/−, Asc^−/−, and Casp1^−/− animals treated with a control siRNA (siRNACtrl.) or siRNAs against Lpin2 (siRNALpin2) for 2 d, and then stimulated as indicated. (right) Level of Lpin2 mRNA after silencing. Data from A–C and E are shown as means ± SD and are representative of at least three independent experiments made in triplicate. Data from D are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.