Figure 3 Effects of TGF-β1 on expression of collagen III and LY3009104 in vivo fibronectin mRNA in HPMCs. Serum-starved HPMCs were incubated with TGF-β1 (2 or 10 ng/ml) for up to 72 h and RNA was then

isolated and subjected to semi-quantitative RT-PCR analysis of collagen III (A) and fibronectin (B). Expression of β-actin was used as a loading control. Figure 4 Western blot analysis of collagen III and fibronectin protein levels in HPMCs with or without TGF-β1 treatment. Serum-starved HPMCs were incubated with increasing concentrations of TGF-β1 for up to 72 h and total cellular protein was extracted and subjected to western blot analysis. A, Dose response of collagen III expression. B, Time course of collagen III expression. C, Dose response of fibronectin expression. D, Time course of fibronectin expression. Figure 5 Confocal immunofluorescence of fibronectin expression in mesothelial cells. Serum-starved RG7112 research buy HPMCs were incubated with TGF-β1 for up to 72 h, and fixed SCH727965 clinical trial for immunostaining with a polyclonal antibody against fibronectin. Fibronectin was visualized by FITC (green), and nuclei were visualized by To-PRO-3 (blue) under immunofluorescence confocal microscopy.

A, Control cells. B, Mesothelial cells treated with TGF-β1 (5 ng/ml) for 72 h. All photos were obtained at 100× magnification. TGF-β1 induction of Smad 2 and 3 phosphorylation in HPMCs To determine how TGF-β1 regulates collagen III and fibronectin expression, we treated HPMCs with 5 ng/ml of TGF-β1; subsequent western blot analysis showed that TGF-β1 induced phosphorylation

of Smad 2 and 3 starting at 10 min post-treatment and reached a maximum between 30-60 min, but TGF-β1 did not affect the total Smad 2 and 3 expression levels (Figure 6). Figure 6 Effects of TGF-β1 on Smad 2 and 3 phosphorylation in the mesothelial cells. The Sitaxentan HPMCs were grown in serum-free medium with or without 5 ng/mL TGF-β1 treatment for up to 24 h. Total cellular protein was then extracted and subjected to Western blot analysis. A, Expression of phosphorylated Smad 2 protein. B, Expression of phosphorylated Smad 3 protein. C, Total Smad 2/3 protein. Induction of gastric cancer cell adhesion to the mesothelial cells through peritoneal fibrosis We then assessed the role of peritoneal fibrosis and RGD (Arg-Gly-Asp sequences) in regulating the adhesion ability of gastric cancer cells to mesothelial cells. Through fluorescently examining the level of tumor cells adhering to mesothelial cells in response to TGF-β1 treatment, we found that peritoneal fibrosis appeared to be able to promote gastric cancer cell adherence to mesothelial cells in a TGF-β1 dose-dependent manner, as compared to the control (p < 0.05). RGD decreased the number of cancer cells to adhere to the mesothelial cells under TGF-β1 stimulation (Figure 7). The data on cancer cells obtained from ascites or no-ascites also showed similar results. Figure 7 Effects of TGF-β1 and RGD on adhesion of gastric cancer cells to mesothelial cells.