TGF-β Signaling Pathway Modulation

Cells were treated with TGF-β (5 ng/ml final; Sigma-Aldrich) for 8 h prior to protein isolation, and for 2 h, 8 h or 24 h prior to RNA isolation, unless noted otherwise. Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment. Actinomycin D (1 ug/ml final; Sigma-Aldrich) treatments were for 1 h after stimulation with TGF-β. Cells were treated with 10 uM final SKF96365 hydrochloride (Sigma, #567310-M) for a period of 24 h prior to TGF-β treatment for 2 h. For treatment with p65 inhibitor ACHP, NMuMG cells were serum starved for 4 hours. After serum starvation, the cells were treated with 2APB for 24 hours. At 18 hours, the cells were treated with 50 uM of the NFKB inhibitor ACHP for a 4-hour pretreatment as previously published [39 (link)] before addition of TGF-β at 22 hours. For the AKT1/2 inhibitor, NMuMG cells were treated with 10 uM of the inhibitor at 20 hours for a 2-hour pretreatment as described previously [38 (link)] before the TGF-β treatment at 22 hours for 2 hours.