Figure 3. BGN inhibits proliferation in human bladder cancer cells in vitro.

(A) Immunoblotting of BGN purified from conditioned media of human J82 bladder carcinoma cells and of recombinant BGN plusminus chondroitinase ABC digestion. As control conditioned medium from human coronary vascular smooth muscle cells (CaSMC) was used because these cells are known to secrete large amounts of BGN proteoglycan (left two lanes). Chondroitinase ABC was applied as additional control (right lane) (B) J82 cells were incubated in low serum (2%) with increasing amounts of recombinant BGN and cell number was determined by FACS analysis after 7 days; n=3. Representative light microscopic images are shown. (C) Human J82 cells were either lentivirally transduced to knock-down BGN expression or to overexpress human BGN. The respective controls were cells transduced with scrambled shRNA (scr) or empty vector control (pCL-1). In addition, recombinant BGN was used. Nine days after transduction, proliferation was determined as evidenced by DNA synthesis. The BGN knock-down had a proliferative effect that was reversed by addition of recombinant BGN (2.6 nM = 100 ng/ml). Overexpression of BGN resulted in reduced DNA synthesis; n=5, *p<0.05.