Impact of lipin-2 on cellular cholesterol levels, and restoration of P2X₇R functionality by cholesterol in the absence of lipin-2. (A) BMDMs from WT and Lpin2^−/− mice (left), or control (siRNACtrl.) and lipin-2–silenced (siRNALpin2) RAW264.7 cells (right) were treated with 200 ng/ml LPS for 4 h, 2 mM ATP for 40 min, or both, as indicated. (B) Total cellular cholesterol levels were analyzed as described in Materials and methods. RAW264.7 cells preincubated with 100 µg/ml cholesterol (cholesterol/MCD) for 30 min were treated as in A, and total cellular cholesterol levels were measured. (C) Example traces of whole-cell ionic currents analyzed by patch-clamp in peritoneal macrophages from WT and Lpin2^−/− animals stimulated with 2 mM ATP in the absence or presence of 100 µg/ml cholesterol (cholesterol/MCD) as indicated. (top) Current density against voltage; (bottom) current densities at −120 and +100 mV. (D) Mean current density from 15–18 cells analyzed. (E) The mean t_on and t_off at −120 and +100 mV from WT and Lpin2^−/− mice peritoneal macrophages stimulated with 2 mM ATP in the presence or absence of 100 µg/ml cholesterol (cholesterol/MCD). Data from A and B are shown as mean ± SD, and experiments shown are representative of at least three independent experiments made in triplicate. Data from D and E are shown as means ± SEM *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test (A and B) or by one-way ANOVA followed by Tukey's test (D and E).