Figure 3

LPS-induced cytokine secretion is altered in DC-STAMP knock-down mBMDCs. Murine BMDCs transduced with shST1, shST4 or shScr were stimulated with 1 μg/ml of LPS for 6, 16 and 24 hours. (A) Levels of secreted IL-6 were measured by ELISA. Data are expressed as a percentage of control (shScr). Mean ± SEM calculated of four independent experiments. Values in all experiments ranged from 5 to 500 ng/ml. (B) Levels of secreted IL-12 p70 were measured by ELISA. Data shown are representative of three independent experiments and are expressed as a percentage of control (shScr). Mean ± SEM calculated from triplicates. Values in all experiments ranged from 0.4 to 5 ng/ml. (C) Levels of secreted cytokines were determined in supernatants 0 and 24 hours after LPS stimulation using Milliplex bead assay. Mean ± SEM calculated from at least three independent experiments. Two-tailed Student t test was performed in A, B and C (ND-not detectable, *p < 0.05; **p < 0.01; ***p < 0.001). (D) Total RNA was isolated from shScr and shST1 mBMDCs stimulated with LPS. Messenger RNA expression levels of IL-6, IL-12p40, IL-1α and IL-1β were determined by quantitative RT-PCR where PBGD served as an internal reference. Results shown are representative of at least three independent experiments. The differences in mRNA levels between shScr and shST1 in all experiments ranged from 20% to 75% for IL-6, 20-65% for IL-12p40, 25-48% for IL-1α and 27-35% for IL-1β.