Inhibition of TGF-β Signaling Pathways

Before stimulation, cells were deprived of serum for 24 h and thereafter stimulated with 1 ng/ml recombinant human TGFβ1 (Biolegend, the Netherlands) for 2 or 24 h. In experiments where inhibitors were used, DMSO was used as vehicle control. To block TAK1 activity, we used (5Z)-7-Oxozeaenol [17 (link)] (Tocris Bioscience) in a concentration of 0.5 μM. To inhibit ALK5 kinase, we used SB-505124 [15 (link)] (Sigma Aldrich) in a concentration of 5 μM. For inhibition of ALK1 kinase, LDN-193189 [14 (link)] (Axon Medchem) was used in a concentration of 0.05 μM. This concentration of LDN-193189 is well above its reported half maximal inhibitory concentration (IC₅₀), 0.8 nM for ALK1, but far below its IC₅₀ for ALK5 of 350 nM [16 (link), 23 (link)]. Cells were pre-incubated with the inhibitors for 1 h prior to addition of TGF-β1. Either 2 or 24 h after addition TGF-β1, medium was removed and TRI-reagent was added for RNA isolation.