Fig. 2
Thrombomodulin enhanced VSMCs adhesion and spreading to type I collagen in a chondroitin sulfate-dependent manner. a HASMCs were infected with lentiviral shRNA constructs targeting luciferase (shLuc) or TM (shTM) for 1-3 days, serum-starved for 2 days, and treated with PDGF for 6 h. TM expression levels were analyzed with Western blotting using β-actin as the loading control. The top panel shows a representative result and the bottom panel summarizes the results of three independent experiments expressed as mean ± SEM. b HASMCs infected with lentivirus carrying luciferase or TM shRNA were seeded on a 96-well plate coated with type I collagen, allowed to adhere for 0.5 h at 37 °C, and stained with crystal violet. Results were obtained from 4 independent experiments and expressed as mean ± SEM, *P < 0.05 compared with shLuc. c and d A7r5 cells transfected with pEGFP, pEGFP-TM, or pEGFP-TMS490,492A were cultured for 36 h. In (c), cells were treated with or without ChABC (0.5 U/ml) for 1 h before adhesion assay. Results were obtained from six independent experiments and expressed as mean ± SEM. In (d), cells were detached, seeded to type I collagen-coated dish for 30 min, and processed for fluorescence microscopy. The left panel shows representative results for cells expressing GFP, TM-GFP, and TMS490,492A-GFP (green) with DAPI nuclear staining; the right panel summarizes the spreading area for each group from at least 3 independent experiments. *P < 0.05, compared with the GFP-expressing group; #P < 0.05, compared with the TM-GFP-expressing group. Scale bar: 100 μm