Gw788388

Primary dermal fibroblasts from ERBB2IP^mut, STAT3^mut, or healthy donors were grown in MEM supplemented with 20% fetal bovine serum and penicillin-streptomycin-glutamine (Thermo Fisher Scientific). 293T cells were cultured in IMDM with 10% FBS and penicillin-streptomycin-glutamine. Transfection of 293T cells with plasmids or siRNAs was accomplished with Nucleofector V (Lonza). Isolated naive primary CD4⁺ T cells were cultured in X-VIVO 15 media (Lonza). Plate-bound anti-CD3 (5 µg/ml unless otherwise indicated; eBioscience), soluble anti-CD28 (1 µg/ml; BD), and IL-2 (20 U/ml unless otherwise indicated; PeproTech) were used in nonskewing and iT reg cell conditions; TGF-β1 (R&D Systems) was used in iT reg cell cultures as indicated. When specified, a neutralizing anti-(α)TGFβ1 antibody (20 µg/ml; R&D Systems), a small molecule ALK5 inhibitor (TGFβR1i) GW788388, or a selective SMAD3 inhibitor (SMAD3i) SIS3 (Sigma-Aldrich) were added at 1 and 5 µM to nonskewing cultures of naive CD4 cells. To test specificity and inhibitory activity on TGF-β pathway activation, these inhibitors were added to the 293T SMAD-reporter line for 12 h before stimulation with TGF-β at the indicated concentrations.