Inhibition of Akt and Smad3 Signaling

To inhibit Akt signaling, cells were seeded in 24-well plates and cultured for 15 min, 30 min, 60 min, 24 h, 3 d, and 7 d in low-serum medium with water and dimethyl sulfoxide (DMSO) (vehicle controls), and low-serum medium supplemented with 50 ng/mL recombinant human TGFβ2 (PeproTech), 500 nM of the Akt inhibitor MK-2206 [36 (link)] (MedChem Express, Monmouth Junction, NJ), or both (TGFβ2 + MK-2206). To inhibit Smad3 signaling, cells were cultured and seeded into 24-well plates as described above for 15 min, 30 min, 60 min, 24 h, 3 d, and 7 d in low-serum medium with water and DMSO (vehicle controls), and low-serum medium supplemented with 50 ng/mL recombinant human TGFβ2 (PeproTech), 2 μM of the Smad3 inhibitor SIS3 [11 (link)] (Tocris, Bristol, UK), or both (TGFβ2 + SIS3).