Figure 2. The effect of lipin-2 on TLR4 signaling. (A) WT and Lpin2−/− BMDMs were stimulated with 200 ng/ml LPS for the indicated periods of time, and then mRNA levels for Tnf were quantified by RT-qPCR. (B) Quantification of TNF present in cellular supernatants from WT and Lpin2−/− BMDMs (left) or silenced human macrophages (right) treated with 200 ng/ml LPS for 4 h. (C) Quantification of Il1b mRNA levels from WT and Lpin2−/− BMDMs (left), or silenced human macrophages (right) stimulated with LPS. (D) Analysis by Immunoblot of pro–IL-1β present in homogenates from BMDMs (left). (right) Relative expression levels of IL-1β against β-actin. (E) Analysis by immunoblot of MAPKs and their phosphorylated forms present in BMDMs activated as in A (left). (right) Relative quantification of phosphoproteins against total proteins. (F) Control (siRNACtrl.) and lipin-2–silenced (siRNALpin2) RAW264.7 cells were pretreated with 10 µM PD98059, SP600125, or SB203580 for 30 min, and then stimulated with 200 ng/ml LPS for 4 h. Il1b mRNA levels were quantified by RT-qPCR. (G) WT and Lpin2−/− BMDMs were treated with inhibitors before or after stimulation with LPS. Stimulations were as indicated. IL-1β present in cellular supernatants is shown. (H) WT and Lpin2−/− BMDMs were stimulated with LPS as in A, and then Nlrp3 mRNA levels were quantified. (I) Analysis by immunoblot of NLRP3 present in cellular homogenates from BMDMs stimulated as in A (left). (right) Relative NLRP3 expression levels against β actin. β-Actin bands are the same as shown in E because the same blot was used. Data from A–C and F–H are shown as mean ± SD, and experiments shown are representative of at least three independent experiments made in triplicate. Data from D, E, and I are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. #, P < 0.05; ###, P < 0.001, versus siRNALpin2 + LPS cells (F), or Lpin2−/− + LPS/ATP cells (G) by Student’s t test.
Figure 2.

The effect of lipin-2 on TLR4 signaling. (A) WT and Lpin2−/− BMDMs were stimulated with 200 ng/ml LPS for the indicated periods of time, and then mRNA levels for Tnf were quantified by RT-qPCR. (B) Quantification of TNF present in cellular supernatants from WT and Lpin2−/− BMDMs (left) or silenced human macrophages (right) treated with 200 ng/ml LPS for 4 h. (C) Quantification of Il1b mRNA levels from WT and Lpin2−/− BMDMs (left), or silenced human macrophages (right) stimulated with LPS. (D) Analysis by Immunoblot of pro–IL-1β present in homogenates from BMDMs (left). (right) Relative expression levels of IL-1β against β-actin. (E) Analysis by immunoblot of MAPKs and their phosphorylated forms present in BMDMs activated as in A (left). (right) Relative quantification of phosphoproteins against total proteins. (F) Control (siRNACtrl.) and lipin-2–silenced (siRNALpin2) RAW264.7 cells were pretreated with 10 µM PD98059, SP600125, or SB203580 for 30 min, and then stimulated with 200 ng/ml LPS for 4 h. Il1b mRNA levels were quantified by RT-qPCR. (G) WT and Lpin2−/− BMDMs were treated with inhibitors before or after stimulation with LPS. Stimulations were as indicated. IL-1β present in cellular supernatants is shown. (H) WT and Lpin2−/− BMDMs were stimulated with LPS as in A, and then Nlrp3 mRNA levels were quantified. (I) Analysis by immunoblot of NLRP3 present in cellular homogenates from BMDMs stimulated as in A (left). (right) Relative NLRP3 expression levels against β actin. β-Actin bands are the same as shown in E because the same blot was used. Data from A–C and F–H are shown as mean ± SD, and experiments shown are representative of at least three independent experiments made in triplicate. Data from D, E, and I are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. #, P < 0.05; ###, P < 0.001, versus siRNALpin2 + LPS cells (F), or Lpin2−/− + LPS/ATP cells (G) by Student’s t test.

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