Effect of lipin-2 on ATP-induced changes in K⁺ cellular levels and cell permeability. (A) Control (siRNACtrl.) and lipin-2–silenced (siRNALpin2) RAW264.7 cells were stimulated with 2 mM ATP for the indicated periods of time, and intracellular concentration of K⁺ was analyzed using an inductively coupled plasma/optical emission spectrometer. (B) RAW264.7 cells were stimulated with 200 ng/ml LPS for 4 h, and then 2 mM ATP for 40 min, as indicated, in the presence of 0, 5, or 45 mM K⁺. IL-1β present in cellular supernatants was analyzed by specific ELISAS. (C) BMDMs from WT and Lpin2^−/− mice were pretreated with 20 µM ethidium bromide (BrEt) for 5 min, and then stimulated with 2 mM ATP. Ethidium cellular uptake was monitored by confocal microscopy. (top) Cell fluorescence; (bottom) fluorescence fold increase over time. Bar, 25 µm. (D) Control (siRNACtrl.) and lipin-2–silenced (siRNALpin2) RAW264.7 cells were stimulated with 2 mM ATP for 50 min in the presence of Annexin-V-Cy3, and fluorescence analyzed in a confocal microscope (top). (bottom) Fluorescence fold increase over unstimulated cells. Bar, 50 µm. Data from A and B are shown as mean ± SD, and experiments shown are representative of at least three independent experiments made in triplicates. Data from C (>100 cells) and D (>300 cells) are shown as means ± SEM, and experiments shown are representative of at least three independent ones. *, P < 0.05; **, P < 0.01; ***, P < 0.001, by Student’s t test.