PMC:3279418 / 10329-12843 JSONTXT

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In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).\n10.1371/journal.pone.0031809.g003 Figure 3 Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.\n(A–D) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours. Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E–F) Gradient numbers (1×104, 2×104, and 4×104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation. After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2]. BMDC developed from cpdm and rescued cpdm mice were plated (1×106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS. After 24 hours, supernatants were collected for analysis of IL12P70 production. Data are representative of three independent experiments. * P\u003c0.01; ** P\u003c0.005.\n10.1371/journal.pone.0031809.g004 Figure 4 Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.\nCultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 µg/ml poly I:C (B,D,F,H). At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA. Bars represent mean ± SD. 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