Fig 1: IL-27 induced LL-37 expression in primary human colonic epithelial cells. (a) Kinetic gene expression of LL-37 in primary human colonic epithelial cells after stimulation with different doses of recombinant human IL-27 protein (20, 50 and 100 μg/ml) for different times (6, 12 and 24 hours). (b) Representative Western blot analysis of LL-37 protein expression in cell lysates of primary human colonic epithelial cells at 24 hour after stimulation with different doses of recombinant human IL-27 protein (20, 50 and 100 μg/ml). Total proteins were extracted from primary human colonic epithelial cells (1 × 106 cells), and an equal amount of protein (10 μg) was subjected to SDS-PAGE (10%) before blotting onto a PVDF membrane. β-Actin was used as a control to ensure an equal amount of loaded protein. (c) Effects of signaling molecule inhibitors on LL-37 protein expression. Primary human colonic epithelial cells were pretreated with AG490 (5 μM; AG), LY294002 (10 μM; LY), SB203580 (20 μM; LY), or U0126 (10 μM; U) for 1 hour, followed by incubation for 24 hour with or without IL-27 (50 ng/ml). LL-37 protein expression in cell lysates was analyzed by Western blot. Densitometry quantification of blots was shown as in histograms on the right. LL-37 expression was normalized to β-actin for each sample, and expression was graphed as fold change above cells at 0 min. Results are expressed as mean ± SD of 5 independent experiments. *P < .05, **P < .01,***P < .001 when compared between groups denoted by horizontal lines