Figure 5: Physical interaction between Tiam1, Rac1 and RORγt and nuclear translocation in murine Th17 cells.
From: Tiam1/Rac1 complex controls Il17a transcription and autoimmunity
(a) Time course of Tiam1/Rac1 interaction as detected by PLA during Th17 cell differentiation. Each red dot represents a single Tiam1/Rac1 molecular interaction. (b) Quantification of the number of Tiam1/Rac1 interactions per cell in the course of Th17 cell differentiation (n=10 cells for each time point). (c) Assessment of the subcellular distribution of Tiam1/Rac1 PLA signals in differentiating Th17 cells. (d) Interaction between Tiam1 and RORγt as detected by PLA at different stages of Th17 cell differentiation. The calibration bars in (a,d) represent 5 μm. (e) Quantification of the number of Tiam1/RORγt interactions per cell during the course of Th17 cell differentiation. (f) Assessment of the subcellular distribution of Tiam1/RORγt PLA signals in Th17 cells. (g) Co-IP analysis of the interaction of Tiam1 with Rac1 and RORγt in fractionated Th17 cells (48 h). Paxillin (cytoplasmic protein) and lamin (nuclear protein) served as controls for the fractionation. (h) ChIP-PCR analysis of Tiam1 and Rac1 binding to the Il17 promoter. Naive CD4+ T cells were stimulated under Th17 cell conditions±NSC23766 for 2 days. Total input DNA before IP was used for normalization of data. The graphs represent quantitative PCR analysis of the ratio of enriched Il17 promoter with RORγt binding sites to the input DNA. (i) ChIP-PCR analysis of Tiam1 and Rac1 binding to the Il17 promoter in naive CD4+ cells isolated from RORγt−/− and control RORγt+/+ mice differentiated for 2 days under Th17 conditions. Data are presented as average±s.e.m. of per cent input with subtraction of control IgG. (j) Il17 luciferase reporter assay. HEK 293T cells were transfected with pGL3-CNS2-Il17 vectors in the presence of the indicated constructs, cells were cultured for 24 h±NSC23766, protein extracts were prepared, and luminescence was measured. Data represent mean±s.e.m. of a representative experiment each performed in triplicate. *P<0.05; **P<0.01 by Student t-test.
