Figure 3.

TRIB3 supports the profibrotic phenotype of AMs by inhibiting A20 activity and increasing C/EBPβ stability. (A) The TRIB3 protein was precipitated from AMs by the anti-TRIB3 antibody. The immunocomplexes were resolved by SDS-PAGE and detected by silver staining. The protein bands were subjected to MS analysis. (B) AMs collected from control subjects were transfected with the Ad-TRIB3 virus. The activity of A20 immunoprecipitated from these cells was measured. (C) The flow cytometric analysis of CD206 expression in AMs from PF mice transfected with the indicated adenovirus. (D) Sample immunoblots demonstrating the expression of C/EBPβ in AMs after transfection with the indicated adenoviruses. (E) Quantitative analysis of C/EBPβ degradation in the presence of Ad-CTRL, Ad-Tnfaip3, or Ad-Tnfaip3 plus Ad-Trib3. (F) Schematic of representation of the construction triple-transgenic mouse model constructed by (i) intercrossing Trib3-targeted knockout mice with homozygous Lyz2^Cre mice to produce Trib3^fl/flLyz2^Cre transgenic mice and (ii) crossing Trib3^fl/flLyz2^Cre mice with Trib3-targeted knockout mice to produce triple-transgenic mice (Trib3^fl/flTnfaip3^fl/+Lyz2^Cre mice. (G–J) Analysis of Masson staining (G), Col1 and C/EBPβ expression in AMs (H), hydroxyproline levels in the right lung (I), and Crs (J) were performed to assess the fibrotic changes in lung tissue after BLM challenge. Scale bar, 200 μm. (B–E) Representatives of 2–4 repeats. (G–J) Representative of two individual experiments. Data are represented as mean ± SEM (n = 5 mice in each group, one-way ANOVA). ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not significant.