Figure 6. The disruption of the IRAK-M homeostatic circuit is due to miR-24-mediated suppression of Smad4.

(a–h) BMMs from C57 BL/6 mice were cultured with M-CSF (10 ng ml⁻¹) in the presence of LPS (0.1 ng ml⁻¹) for 5 days and SP600125 or miR-24 antagomir was added to indicated cultures. Fresh LPS, SP600125 and miR-24 antagomir was added every 2 days. The mRNA and protein levels of SMAD4 and IRAK-M in the cell cultures were analysed by real-time reversre transcriptase–PCR and western blotting, respectively. (i) A schematic illustration of the SMAD4 3′-UTR and potential miR-24 binding sites. (j) Luciferase activity assays in 293T cells transfected with SMAD4 3′-UTR luciferase reporter plasmids in the presence of miR-24 mimic or the scramble control. (k) BMMs were cultured for 5 days and miR24 antagomir was transfected. On 7 days, the cells were stimulated by LPS (0.1 ng ml⁻¹) for 4 h and then harvested for RNA co-immunoprecipitation (RIP). Quantified data are shown from cells with indicated treatment (n=3) and results are representative of three experiments. Error bars show means±s.e.m.; NS, not significant; *P<0.05, **P<0.01 and ***P<0.001; one-way analysis of variance.