Figure 3. Ash1l enhances TGF-β-Smad2/3 signalling by promoting Smad2/3 expression.

(a) mRNA expression of Smad2 (left) and Smad3 (right) in the WT and Ash1l-silenced CD4⁺ T cells stimulated under iTreg cell-skewing conditions (with TGF-β) for indicated times. Results are relative to those in unstimulated WT CD4⁺ T cells, set as 1. (b) ELISA assay of Smad2/3 in 5 μg whole-cell extract (left) and 5 μg nuclear extract (right) of WT and Ash1l-silenced CD4⁺ T cells under iTreg cell-skewing conditions (with TGF-β) for indicated times. OD₄₅₀ represents absorbance at 450 nm. (c) mRNA expression of Smad2 and Smad3 in the WT and Ash1l-silenced CD4⁺ T cells transduced with a control lentivirus (Lenti-CTR) or Ash1l-fragment-expressing lentivirus (Lenti-Ash1l-F1, Lenti-Ash1l-F2, Lenti-Ash1l-F3 or Lenti-Ash1l-ΔN) and cultured under iTreg cell-skewing conditions (with TGF-β) for 3 days. Results are relative to those in WT CD4⁺ T cells transduced with Lenti-CTR, set as 1. (d) ELISA assay of Smad2/3 in 5 μg whole-cell extract (left) and 5 μg nuclear extract (right) of WT and Ash1l-silenced CD4⁺ T cells transduced and cultured as in c. OD₄₅₀ represents absorbance at 450 nm. Error bars represent s.d. Student's t test. *P<0.05, **P<0.01. All data are from three independent experiments (mean±s.d. of technical triplicates).