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. 2014 Jul 29;143(1):81–95. doi: 10.1111/imm.12296

Figure 8.

Figure 8

EP4 receptor of ptostaglandin E2 (PGE2) is involved in the anti-inflammatory effect of docosahexaenoic acid (DHA). (a) RAW264.7 cells were pre-treated with an EP2 inhibitor, AH6809 (3 μm), or an EP4 inhibitor, GW627368X (2 μm) for 1 hr, then stimulated with 100 μm DHA for 1 hr and finally stimulated with 10 ng/ml lipopolysaccharide (LPS) for 6 hr. Cell culture supernatants were collected and assayed for IL-6 secretion. Data shown are the mean ± SEM (n = 3) of IL-6 secretion (pg/ml) and are representative of the results of three independent experiments. **P < 0·01 as indicated, as assessed by analysis of variance with Holm–Sidak post hoc test, ND = non-detectable. (b) Knock-down efficiency of EP2 or EP4 siRNA. RAW264.7 cells were transfected with EP2 siRNA (100 nm), EP4 siRNA (100 nm) or Negative Control siRNA (100 nm). Total RNA was isolated from transfected cells. The mRNA level of EP2 and EP4 expression was determined by Taqman real time PCR. The results are presented as the mean ± SEM (n = 3) of percentage change compared to negative control siRNA. ##P < 0·01 as indiated, as assessed by Student's t-test. The protein level of EP2 and EP4 was detected by Western blot and was quantified. Data are representative results of three independent experiments, each showing similar results. (c) RAW264.7 cells were transfected with either EP2 siRNA (100 nm), EP4 siRNA (100 nm) or Negative Control siRNA (100 nm) for 48 hr, stimulated with 100 μm DHA for 1 hr, then stimulated with lipopolysaccharide (LPS; 10 ng/ml) for 6 hr. Cell culture supernatants were assayed for IL-6 secretion. Data are presented as the mean ± SEM (n = 3) of IL-6 secretion (pg/ml) and are representative of the results of three independent experiments. **P < 0·01 as indicated, as assessed by analysis of variance and Holm–Sidak post hoc test.