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TRIM32-TAX1BP1-dependent selective autophagic degradation of TRIF negatively regulates TLR3/4-mediated innate immune responses

Fig 5

TRIM32 promotes TAX1BP1-mediated selective autophagic degradation of TRIF.

(A) Effects of different inhibitors on TRIM32-mediated destabilization of TRIF. HEK293 cells were transfected with the indicated plasmids for 20 hours and then treated with the indicated inhibitors for 6 hours before immunoblotting analysis with the indicated antibodies. (B) Effects of poly(I:C) treatment on autophagy. HEK293-TLR3 cells were transfected with a GFP-tagged expression plasmid for LC3. Twenty hours after transfection, cells were treated with poly(I:C) (50 ng/mL) for 3 hours and subjected for confocal microscopy. (C) Effects of poly(I:C) treatment on colocalization of TRIF and LC3. HEK293-TLR3 cells were transfected with the indicated plasmids. Twenty hours after transfection, cells were treated with poly(I:C) (50 ng/mL) for 3 hours before subjected for confocal microscopy. (D) Effects of 3MA and MG132 on poly(I:C)-induced degradation of TRIF. Trim32+/+ and Trim32-/- BMDMs were pre-treated with 3MA or MG132 for 2 hours, followed by poly(I:C) stimulation for the indicated times before immunoblotting analysis was performed. (E) Effects of double deficient of TRIM32 and TRIM38 on poly(I:C)-induced degradation of TRIF. Trim32+/+ and Trim32-/- MLFs stably transduced with control or TRIM38-RNAi plasmids were left untreated or treated with poly(I:C) (50 μg/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (F) Knockdown of LC3B inhibits TRIM32-mediated degradation of TRIF. HEK293 cells were transfected with a RNAi control or LC3B-RNAi plasmid for 24 hours, and then further transfected with the indicated plasmids for 24 hours before immunoblotting analysis with the indicated antibodies. (G) Effects of TRIM32 on interaction of TRIF with LC3B. HEK293 cells were transfected with the indicated plasmids for 24 hours before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (H) Effects of TRIM32 on the conversion of LC3B-I to LC3B-II. HEK293 cells were transfected with the indicated plasmids for 24 hours before coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. (I) Effects of ATG7-deficiency on poly(I:C)- or LPS-induced degradation of TRIF. Atg7+/+ and Atg7-/- MEFs (2x106) were treated with poly(I:C) (50 μg/ml) or LPS (50 ng/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. (J) Endogenous association of TRIM32 with TRIF. MLFs were pre-treated with bafilomycin for 2 hours, followed by poly(I:C) stimulation for the indicated times before coimmunoprecipitation was performed.

Fig 5

doi: https://doi.org/10.1371/journal.ppat.1006600.g005