Fig. 4.
Differential modulation of SMAD2/3 inhibits RORγt-mediated Il17a transcription by forming complex with RORγt. (A) Flow cytometry of RORγt staining from naïve CD4+ T cells primed under TH17 polarizing condition for 2 d. (B) RORα was immunoblotted with whole-cell lysate of TH17 cells. (C and D) Flow cytometry (C) and quantification (D) of TH17 polarization with ectopic expression of Vector or RORγt in PP2A WT and cKO naïve CD4+ T cells. GFP-expressing cells were gated for analysis on day 3. (E) RORγt binding to the sites of the Il17a gene promoter was analyzed by using chromatin immunoprecipitation (ChIP) assay with RT-PCR. (F) Co-IP analysis of binding ability with RORγt among SMAD2/3 in WT and cKO TH17 cells. (G) Co-IP analysis of binding ability with RORγt among WT, 2SA, and 2SD mutant of SMAD2/3 in 293FT cells. (H) Flow cytometry of TH17 polarization with ectopic expression of Vector or SMAD2-2SD in PP2A WT and cKO naïve CD4+ T cells. (I) Ratio of IL17A frequency comparing VEC-cKO or SMAD2-2SD-cKO to VEC-WT. (J) Flow cytometry of TH17 polarization after suppressing SMAD3 expression by siRNA in naïve CD4+ T cells. (K) The ratio of IL17A frequency in cKO cells transfected with siRNA-SMAD3 or control to the frequency of WT cells transfected with control. Error bars show mean ± SEM. Data are representative of three (A, B, G, H, and J) or two (C–F) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.