Figure 2.

SIK inhibition suppresses IL-33–dependent production of il13, gm-csf, and tnf mRNA in BMMC.A–C, The experiment was performed as in Figure 1 except that, at the times indicated, cells were lysed and the mRNA encoding IL-13 (A), GM-CSF (B), and TNF (C) measured as described in Experimental procedures. The figures show the mRNA level relative to gapdh mRNA and are presented as the mean and standard deviation of four biological replicates. The results using HG-9-91-01 and MRT199665 are from the same experiment but plotted in separate panels for clarity, and similar results were obtained in two independent experiments. Statistical analysis is represented by repeated measures two-way ANOVA with Dunnett’s multiple comparison test, comparing each inhibitor to control; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. D–F, WT BMMCs were incubated for 2 h with 10 ng/ml IL-33. Cells were then treated with 50 μM DRB and 5 μg/ml actinomycin D to block transcription without (control, open circles) or with 0.5 μM HG-9-91-01 (closed circles, left panel), 3 μM MRT199665 (closed triangles, right panel), or 1 μM VX745 (closed squares, right panel). At the times indicated, cells were lysed, and the il13 (D), gm-csf (E), and tnf (F) mRNA levels were measured, normalized to gapdh mRNA, and plotted relative to mRNA levels measured at the point of transcription inhibition. The results are presented as the mean and standard deviation of three replicates. Similar results were obtained in three independent experiments. The results using HG-9-91-01, MRT199665 and VX745 are from the same experiment, but plotted in separate panels for clarity. DRB, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside; BMMC, bone marrow-derived mast cell; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; SIK, salt-inducible kinase; TNF, tumor necrosis factor.