Figure 4. P529 inhibits TGF-β-induced actin stress fiber formation and fibronectin deposition into the ECM.

HLF were treated with 1 ng/ml TGF-β for 24 h and P529 or vehicle control, as indicated. A) Cells were then subjected to staining with phalloidin (red) and DAPI (Blue) and visualized with a 60x objective. Scale bar = 50 μm. B) Cells were stained with anti-fibronectin antibody (green) and DAPI (Blue) and visualized with a 10x objective (top row) or a 60x objective (bottom row). Scale bar = 250 μm. C, D) HLF were transfected with siRNA targeting mTOR or scrambled control, followed by treatment with 1 ng/ml TGF-β for 24 h, followed by western blotting and densitometry of the indicated proteins. Statistical significance was tested using student’s t-test. * denote statistically significant differences (p<0.05) between conditions. E) HLF were plated and grown overnight on tissue culture plastic. Serum-free media with DMSO or 10 μM P529 was then added to induce growth arrest. Linear wounds were made at the time of serum removal and imaged. Cells were fixed after 24 h and imaged with a 10x objective. Images shown have been processed with ImageJ to enhance edges and contrast for clarity. F) Closure after 24 h was measured using Image J. Scale Bar = 200 μM. Statistical significance was tested using student’s t-test. * denote statistically significant differences (p<0.05) between conditions.