Figure 7.

RSK regulated IL-10 production in LPS–stimulated B cells. A, peritoneal cavity cells from wildtype mice were incubated with 10 μm LJI308 for 1 h prior to stimulation with 10 μg/ml LPS + brefeldin A + monensin for 5 h. Intracellular IL-10 in B cells and macrophages was assessed by flow cytometry. Representative plots are shown in the left panels. The data shown in the right panel represent the averages ± standard deviations of biological replicates (n = 4). ***, p value < 0.001 compared with LPS–stimulated cells (two-tailed unpaired Student's t test). B, peritoneal cavity cells from wildtype, RSK1 knockout (KO), RSK2 knockout, and RSK1/2 double knockout (DKO) mice were stimulated with 10 μg/ml LPS + brefeldin A + monensin for 5 h. Intracellular IL-10 in B cells and macrophages was assessed by flow cytometry. Representative plots are shown. C, quantification of data from B. The graphs represent the averages ± standard deviations of biological replicates (n = 13, 5, 8, and 4 for LPS–stimulated WT, RSK1, RSK2, and RSK1/2, respectively). One-way ANOVA for LPS–treated samples showed a that the effect of genotype on the percentage of IL-10–positive cells was significant in B cells (F(3,26) = 10.745, p < 0.001) but not macrophages (F(3,26) = 0.128, p = 0.943). For pairwise comparisons with wildtype cells using the Holm–Sidak method, * indicates p < 0.05, and ** indicates p < 0.001. D, B cells were purified form peritoneal washes from wildtype mice as described under “Materials and methods.” The cells were incubated with 10 μm LJI308 for 1 h prior to stimulation with 10 μg/ml LPS for 2h. Total RNA was isolated, and induction of IL-10 mRNA and IL-10 primary (1⁰) transcript was determined by qPCR relative to unstimulated cells. Graphs show means ± standard deviations (n = 5). **, p < 0.01; ** p < 0.001 compared with LPS–stimulated cells (two-tailed Student's t test). Con, control.