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Figure 2: 4-PG decreases LPS-induced proinflammatory cytokine expression via enhancement of TTP in RAW264.7 cells. RAW264.7 cells were pretreated with 4-PG (10 μM) for 6 h followed by the stimulation of LPS (100 ng/ml) for 4 h. (a) The mRNA expression of IL-6, TNF-α, and TTP was measured by RT-PCR. (b, c) The secreted protein level of IL-6 and TNF-α in the supernatant of cultured cells was determined by ELISA. (d) Effects of 4-PG on reactive oxygen species (ROS) production were detected in RAW 264.7 cells. RAW264.7 cells were preincubated with or without 4-PG (10 μM) for 6 h and N-acetyl-L-cysteine (NAC, 1 mM) for 30 min followed by the stimulation of LPS (100 ng/ml) for another 4 h. The intracellular level of ROS was stained by H2DCFDA, and the production of ROS was measured by flow cytometry. The fold change of fluorescence intensity is presented as means ± SE, . . To confirm that the anti-inflammatory effect of 4-PG was mediated by TTP, RAW264.7 cells were transfected with scramble RNA (scRNA) and siRNA against TTP (siTTP) for 24 hours. Then, cells were pretreated with 4-PG (10 μM) for 6 h followed by the challenge of LPS (100 ng/ml) for 4 h. (e, f) The mRNA expression of TTP and TNF-α was analyzed by real-time PCR, respectively. GAPDH was used as internal control. (g, h) To determine the mRNA-decaying activity of TTP on inflammatory cytokines, bone marrow-derived macrophage (BMDM) extracted from Ttp+/+ and Ttp−/− mice was pretreated with 4-PG (10 μM) for 6 h followed by the stimulation of LPS (100 ng/ml) for 4 h. Then, the mRNA level of IL-6 (g) and TNF-α (h) was measured by real-time PCR at indicated times (0, 30, 60, and 120 min) after the addition of 5 μg/ml actinomycin D. Data are expressed as means ± SE, . , . ns: not significant.