Supplementary Figure 7: TEAD sequesters TAZ from the T_H17 master regulator RORγt to promote the development of T_reg cells.

(a) Immunoassays of 293T cells expressing various combinations (above lanes) of Flag-tagged RORγt, Foxp3 or TEAD1 and HA-tagged TAZ; immunoprecipitation with anti-Flag and analysis by immunoblot with the indicated antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. (b) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of Flag-tagged TAZ^WT or TAZ^S51A, GFP-tagged TEAD1 or increasing doses of GFP-tagged RORγt. (c) SIM of HeLa cells cotransfected with NLS/Flag-tagged TAZ^WT or TAZ^S51A (purple), HA-tagged TEAD1 (red) or GFP-tagged Foxp3 (green); outlined areas are enlarged 4× in the corner insets. Scale bars, 20 μm. (d) Immunoassays (as in a) of 293T cells expressing various combinations (above lanes) of Flag-tagged TAZ, HA-tagged Tip60 or increasing doses of HA-tagged TEAD1. (e) Immunoassay (as in a) of 293T cells expressing various combinations (above lanes) of HA-tagged Tip60, HA-tagged p300, Flag-tagged Foxp3 and/or GFP-tagged TAZ or HA-tagged TEAD1, treated with DMSO or the histone deacetylase inhibitors Trichostatin A and Nicotinamide (TSA&NAM). immunoprecipitation with anti-Flag and analysis by immunoblot with anti-K-Ace (α-K-Ace), anti-Flag (α-Flag), anti-HA (α-HA) or anti-GFP (α-GFP); below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation.

(f) Immunoblot analysis of TEAD1 in CD4⁺ naive T cells infected with a retrovirus expressing shRNA for knocking down TEAD1 or control shRNA, differentiated under Treg -polarizing conditions. The data are representative of three independent experiments with similar results (a-f), or with ~50 cells (c).