TAK1 controls the activation of multiple transcriptional factors relevant for the induction of EMT and fibrosis.

<p><b>A</b>, Effect of TAK1 inhibition on NF-κB transcriptional activity. MeT-5A cells were transiently transfected with the KBF-luc reporter plasmid together with Renilla luciferase. Cells were pretreated with NP-009245 (600 nM) for 12 h and then left untreated (NT, gray bars) or co-stimulated for 9 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Bars represent means+s. e. m. of triplicate determinations in three independent experiments. <b>B</b>, Effect of TAK1 inhibition on PAI-1 transcriptional activity. MeT-5A cells were transiently transfected with a PAI-1 reporter plasmid together with a Renilla luciferase-coding plasmid. Cells were stimulated as above. Bars represent means+s. e. m. of triplicate determinations in three independent experiments <b>C</b>, Western blots showing the expression of phospho-Smad3, phospho-Smad1–5, PAI-1 and phospho-c-jun in total cell lysates of HPMCs treated for different times with TGF-β1 0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Expression of tubulin was used as a loading control. The results shown are representative of three independent experiments. <b>D</b>, Effect of TAK1 inhibition on Smad1–5–8 transcriptional activity. MeT-5A cells were transiently transfected with the BRE-luc reporter plasmid together with a Renilla luciferase-coding plasmid. Cells were pretreated with NP-009245 (600 nM) for 12 h and then left untreated (NT, gray bars) or co-stimulated for 12 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (T/I). Bars represent means+s. e. m. of triplicate determinations in three independent experiments. <b>E</b>, Western blots showing the expression of phospho-Smad1–5, PAI-1 and TAK1 in whole cell lysates of HPMCs transfected with either control or specific TAK1-targeting siRNAs and then left untreated or stimulated for 4 h with TGF-β1 (0.5 ng/ml) in combination with IL-1β (2 ng/ml) (TI). Scr: cells transfected with control siRNA. Data are representative of three independent experiments <b>F</b>, Western blot showing the expression of phospho-Smad1–5–8 in MCs from a patient undergoing PD. Cells were treated with DMSO, CI 1040 (2 µM), NP-009245, (600 nM) for 24 h. Expression of tubulin was used as a loading control. All data are representative of at three independent experiments. * p<0.05 versus corresponding sample treated with DMSO.