Figure 5: Lnc-Smad3 inhibits iTreg cell polarization via suppressing Smad3 transcription.

(a) A schematic outlining the genomic loci of lnc-Smad3 and Smad3. Semi-quantitative PCR (b) and gel electrophoresis (c) detection of lnc-Smad3 in CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. Results are relative to the baseline lnc-Smad3 expression in unstimulated CD4⁺ T cells, set as 1. (d) Relative expression of lnc-Smad3 in WT and Smad3-KO CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to the lnc-Smad3 expression in unstimulated WT CD4⁺ T cells, set as 1. (e) CHIP analysis of the accumulation of Smad3 at the lnc-Smad3 promoter regions with anti-Smad3 antibody in CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for 2 days. Normalized data are shown as percentage of input control (% Inp). IgG serves as a CHIP control. (f) Relative expression of lnc-Smad3, Smad3 and Foxp3 in CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or lnc-Smad3-expressing lentivirus (Lenti-lnc-Smad3) and cultured under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to those in CD4⁺ T cells transduced with Lenti-CTR, set as 1. (g,h) The percentages of Foxp3⁺ Tregs in CD4⁺ T cells transduced and cultured as in f were analysed by flow cytometry (g) and quantified (h). Numbers in quadrants indicate per cent cells in each. Error bars represent s.d. Student’s t test. NS, not significant. *P<0.05, **P<0.01. Data are from three independent experiments (b,d-f,h; mean±s.d. of technical triplicates) or are representative of three independent experiments (c,g).