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. 2010 Oct 18;120(11):3979–3995. doi: 10.1172/JCI42315

Figure 6. The activity of the Rho/ROCK/NF-κB pathway is inhibited in S1pr2-deficient macrophages.

Figure 6

(A–C) Serum-starved peritoneal macrophages from S1pr2+/+Apoe–/– (white bars) and S1pr2–/–Apoe–/– (black bars) mice were stimulated with S1P (0.1 μM) for 2 minutes (A), 5 minutes (B), or 20 minutes (C) and analyzed for GTP-Rho (A), phosphorylation (Thr850) of MYPT1 (B), and phosphorylation (Ser536) of NF-κB (p65) (C) as described in Methods (n = 3 each). (D) Peritoneal macrophages were stimulated with S1P (0.1 μM) for 6 hours. TNF-α mRNA level was determined by real-time PCR. (E–G) BM-derived macrophages from S1pr2+/+Apoe–/– (white bars) and S1pr2–/–Apoe–/– (black bars) mice were cultured in DMEM containing 10% FBS and treated with either JTE-K1, Y-27632 (Wako), or BAY11-7085 (Merck-Calbiochem) for 6 hours. mRNA expression levels of CD36 (E), ABCA1 (F), and LXRα (G) were determined by real-time PCR (n = 5 each). (H) Foam cell formation assay. Serum-starved BM-derived macrophages from S1pr2+/+Apoe–/– mice were exposed to oxLDL for 6 hours. S1P (0.1 μM), JTE-K1, Y-27632, and/or BAY11-7085 were added 6 hours before adding oxLDL. Quantified data of oil red O–positive cells are shown (n = 3 each). In A–H, the inhibitors (10 μM JTE-K1, 10 μM Y-27632, and 10 μM BAY11-7085) were added 10–30 minutes before S1P addition and present in the medium throughout the subsequent incubation. *P < 0.05; **P < 0.01; ***P < 0.001, as compared with nontreated S1pr2+/+Apoe–/– macrophages. #P < 0.05; ##P < 0.01; ###P < 0.001, as compared with S1P-treated S1pr2+/+Apoe–/– macrophages. Data are expressed as the ratio of the values in treated cells over nontreated cells and are shown as mean ± SEM (n = 5 each).