HILI Inhibits TGF-β Signaling by Interacting with Hsp90 and Promoting TβR Degradation
Figure 1
Effects of HILI on TGF-β signaling at the level of Smad phosphorylation and ultimate effect on apoptosis.
A. HILI reduces expression of, PAI-1, and Smad7 and prevents Smad2/3 activation. HEK-293 cells were transfected with Myc-HILI at a concentration gradient (0.5 µg, 0.5 µg, 1.5 µg, and 3 µg per well) and treated with TGF-β for 1 h before cells were harvested as indicated. Cells lysates were used for Western blot analysis with anti-, anti-PAI-1, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2/3, anti-Smad4, anti-Smad7, anti-GAPDH, and anti-Myc antibodies. B. siHILI reverses the reduction of p21, PAI-1, Smad7, and phosphorylated Smad2/3 mediated by HILI. HEK-293 cells were transfected with siHILI or Myc-HILI, after 48 h, treated with TGF-β for 1 h before harvesting. They were then used in Western blot analysis by using antibodies as in (A). NC represents negative control transfected with siControl. C and D. HILI inhibits TGF-β-induced cell apoptosis. HEK-293 cells were respectively transfected with pcDNA3.1, Myc-HILI, siControl and siHILI, and treated with TGF-β for 12 h before cells were harvested as indicated. After transfection 48 h, apoptosis of cells was analyzed with Annexin V/PI double staining and flow cytometry. NC represents negative control without any treatments. Each experiment was performed in triplicate. *indicates P<0.05.