TRIM32-deficiency potentiates TLR3/4-mediated signaling.

(A) Effects of TRIM32-deficiency on poly(I:C)-, LPS-, PGN-, or R848-induced transcription of Ifnb1, Isg56, Tnfa and Il6 in BMDMs, BMDC and MLFs. Trim32^+/+ and Trim32^-/- cells were stimulated with poly(I:C) (50 μg/ml), LPS (50 ng/ml), R848 (20 nM) or PGN (20 μg/ml) for the indicated times before qPCR was performed. (B) Effects of TRIM32-deficiency on poly(I:C)- and LPS-induced phosphorylation of IRF3 and IκBα in MLFs. Wild-type and Trim32^-/- MLFs were stimulated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) for the indicated times before immunoblots were performed with the indicated antibodies. (C) Effects of TRIM32-deficiency on MPLA-induced transcriptions of Ifnb1, Isg56, Tnfa and Il6 genes in BMDMs. Trim32^+/+ and Trim32^-/- BMDMs were stimulated with MPLA (100 ng/ml) for the indicated times before qPCR was performed. (D) Effects of TRIM32 on poly(I:C)- or LPS-triggered activation of IFN-β, ISRE and NF-κB. HEK293-TLR3 cells or HEK293-TLR4 cells (1x10⁵) were transfected with the indicated luciferase plasmid (0.1 μg) and an HA-tagged expression plasmid for murine TRIM32 (0.05 μg). Twenty hours after transfection, cells were treated with poly(I:C) (50 ng/mL) or LPS (100 ng/ml) for 10 hours before luciferase assays were performed. (E) Effects of TRIM32 on IFNγ-induced activation of the IRF1 promoter. HEK293 cells were transfected with the IRF1 promoter reporter plasmid (0.1 μg) and the indicated amounts of HA-tagged murine TRIM32 expression plasmids (0.05 μg). Twenty hours after transfection, cells were treated with IFNγ (100 ng/mL) or left untreated for 10 hours before luciferase assays were performed. n.s., Not Significant. *, p<0.05; **, p<0.01.