Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis
J. Clin. Invest. Yingjian Li, et al. 112:503 doi:10.1172/JCI17913 [Go to this article.]

Figure 2
ILK induction by TGF-β1 in renal epithelial cells is dependent on Smad signaling. (a–c) Pharmacological inhibition of different signal transduction pathways does not affect ILK induction by TGF-β1. HKC cells were pretreated with either various chemical inhibitors or vehicle (DMSO) for 30 minutes, followed by incubating in the absence or presence of 2 ng/ml TGF-β1 for 0.25, 0.5, 1 hour, 3 hours (a and b) and for 24 hours (c), respectively. Specific inhibitors for PI3K (10 nM wortmannin), Mek1 (10 μM PD98059), p38 MAPK (20 μM SC68376), PKA (0.3 μM PKA inhibitor [PKAI]), and PKC (50 nM Ro-31-8220) were used, respectively. Cell lysates were immunoblotted with Ab’s against phosphospecific Akt (p-Akt) and total Akt (a), phosphospecific and total p38 MAPK (p-p38 and p38, respectively) (b), ILK, and actin (c), respectively. (d–f) Overexpression of inhibitory Smad-7 abolishes ILK induction by TGF-β1. A stable cell line overexpressing inhibitory Smad-7 (HKC^Smad7) was established by transfection of Smad-7 expression vector. A cell line with mock transfection of empty pcDNA3 vector (HKC^pcDNA3) was used as control. Cells were treated with 2 ng/ml of TGF-β1 for various periods of time as indicated. (d) Cell lysates were blotted with phosphospecific (p-Smad2) and total Smad-2, respectively. (e) Cell lysates were blotted with Ab’s against ILK, Smad-7, and actin, respectively. (f) Graphical presentation of relative ILK abundance normalized to actin following TGF-β1 treatment in HKC^pcDNA3 and HKC^Smad7 cells. AU, arbitrary units.