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. 2018 Feb 16;159(4):1644–1657. doi: 10.1210/en.2017-03127

Figure 7.

Figure 7.

TβRII/ALK5-mediated SMAD signaling is required for TGF-β1–induced upregulation of SNAIL in SVOG cells. (A) A total of 5 µg/mL rTβRII was preincubated with 10 ng/mL TGF-β1 at room temperature for 1 hour and was then added to cells for 24 hours. SNAIL mRNA levels were examined using RT-qPCR. (B and C) Cells were treated with 10 ng/mL TGF-β1 for 24 hours in the presence of vehicle control [dimethyl sulfoxide (DMSO)], 10 μM SB431542 or 10 μM dorsomorphin dihydrochloride. SNAIL (B) mRNA and (C) protein levels were examined using RT-qPCR and western blot analysis, respectively. (D and E) Cells were transfected with siRNAs (siControl, siALK4, or siALK5) for 48 hours and then treated for 6 hours with vehicle control or 10 ng/mL TGF-β1. SNAIL (D) mRNA and (E) protein levels were examined using RT-qPCR and western blot analysis, respectively. (F) Cells were transfected for 48 hours with 25 nM control siRNA (siControl), SMAD2 siRNA (siSMAD2), SMAD3 siRNA (siSMAD3), or both SMAD2 siRNA and SMAD3 siRNA (siSMAD2+siSMAD3) and then treated with 10 ng/mL TGF-β1 for an additional 6 hours. SNAIL mRNA levels were examined using RT-qPCR. (G and H) Cells were transfected for 48 hours with 25 nM siControl or siSMAD4 and then treated with 10 ng/mL TGF-β1 for an additional 6 hours. SNAIL (G) mRNA and (H) protein levels were examined using RT-qPCR and western blot analysis, respectively. The results are expressed as the mean ± standard error of the mean of at least three independent experiments; lowercase letters indicate statistically significant differences (P < 0.05). C, control; Ctrl, Control; DM, dorsomorphin dihydrochloride; SB, SB431542; T, TGF-β1.