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. 2023 Sep 27;120(40):e2215421120. doi: 10.1073/pnas.2215421120

Fig. 6.

Fig. 6.

TGFβ1-mediated antagonism of IL-27p28 release via SMAD3, p38, TTP, and IL-10. (A) IL-27p28 release with reduced activity of TGFβ1 in lentiviral transduced RAW264.7 macrophages with shRNA knockdown of SMAD3 (two independent shRNAs) as compared to nontarget shRNA (Ctrl), 24 h, ELISA. (B) TGFβ1 partially fails to suppress il-27p28 mRNA expression in transduced RAW264.7 macrophages after treatments with LPS ± TGFβ1, 6 h, RT-PCR. (C) TGFβ1 restores SMAD3 binding to the il-27p28 promoter region, BMDM, 3 h, ChIP assay. (D) TGFβ1 restores SMAD3 binding to the ebi3 promoter region, BMDM, 3 h, ChIP assay. (E) Effects of several p38 MAPK inhibitors (SB 203580 [10 µM], PH-797804 [10 nM], BIRB796 [50 nM]) on IL-27p28 release in BMDM after LPS ± TGFβ1, 24 h, ELISA. (F) Reduced capacity of TGFβ1 to suppress IL-27p28 release in BMDM from tristetraprolin-deficient (TTP−/−) mice as compared to WT mice, ELISA, 24 h. (G) Flow cytometry of F4/80+CD11b+IL-27p28+ BMDM from TTP−/− mice and WT mice after LPS ± TGFβ1, 12 h, pregated on CD11b. (H) IL-27p28 release from BMDM incubated with LPS ± TGFβ1 in the copresence of blocking IL-10 receptor antibodies (α-IL10R; 10 µg/mL) or rat IgG1 isotype control antibodies (10 µg/mL), ELISA. (I) Defective suppression of IL-27p28 by TGFβ1 in BMDM from IL-10−/− mice as compared to WT mice. BMDM were from C57BL/6 J (WT) mice in all frames unless specified otherwise; LPS (100 ng/mL), TGFβ1 (10 ng/mL). Data in each frame are representative of three independent experiments; Student’s t test or one-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns: not significant.