Fig. 5

TGF-β promotes AREG expression in Treg Cells through activation of Smad3. (A-B) Spleen CD4⁺ T cells were isolated and cultured Treg-polarization conditions with or without the FOXP3 inhibitor. (A) Tregs ratio was determined by flow cytometry. (B) Areg expression was detected by qRT-PCR. (C-D) Spleen CD4⁺ T cells were isolated and cultured with or without 10 ng/ml IL-2, 10 ng/ml IL-10 or 2 ng/ml TGF-β for 5 days. (C) Areg expression was detected by western blot and (D) qRT-PCR. (E) Spleen CD4⁺ T cells were isolated and cultured with 2ng/ml TGF-β for 1, 2, 3, and 5 days. Areg expression was detected by qRT-PCR. (F) Spleen CD4⁺ T cells were isolated and cultured with TGF-β at indicated dose for 5 days. Areg expression was detected by qRT-PCR. (G) CD4⁺ T cells were cultured with TGF-β with or without TGF-β receptor inhibitor. Areg expression was detected by qRT-PCR. (H) CD4⁺ T cells were cultured with or without TGF-β for 12 h. Phosphorylated Smad3 and total Smad3 were detected by western blot. (I-J) CD4⁺ T cells were cultured with or without TGF-β and Smad3 inhibitor for 12 h. Phosphorylated Smad3, total Smad3 and AREG were detected by western blot. Representative data from 2–3 independent experiments with similar results. (A, B, D, G and H) Unpaired Student’s t-test; (C, I and J) one-way ANOVA. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001