Fig. 3

ACC cells were either treated with exogenous recombinant DKK3 (a) or enforced to express Myc-DDK tagged DKK3 (b) and assayed for cell behaviors. a SW-13 N/D (left) or NCI-H295R (right) cells were untreated (SW N/D-, 295-) or treated (SW N/D+, 295+) with exogenous DKK3 for 24 h and allowed to migrate through modified Boyden chamber for 4 h. Cells migrating to lower surface were fixed, stained, and counted. b Western immunoblot detection of endogenous DKK3 and ectopically expressed DKK3 (Myc-DDK/DKK3) in vector control (lane 1), SW-DKK3 (lane 2), or Myc-DDK/GFP control (lane 3) cells. c SW-13, SW-Neo, and SW-DKK3 cells plated in 24-well plates (5000 cells/well) were grown 8 days. Quadruplicate wells from each cell type were trypsinized, incubated in 0.2% Trypan blue, and viable cells were counted using hemocytometer. Data shown represent one of three independent experiments. d and e, Five thousand SW-13 or SW-DKK3 cells plated in 6-well plates were allowed to grow 7 days; clones were fixed, stained, and enumerated into 2 classes of (a) 12 ± 2 cells (filled light grey) and (b) 4 ± 2 cells (filled black). Majority of clones formed from SW-Neo cells were large (e; left), while SW-DKK3 cells produced a significant number of small colonies (4 ± 2) comprised of large cells (e; right). f One hundred thousand SW-13, SW-Neo, and SW-DKK3 cells were allowed to migrate through modified Boyden chamber for 4 h; cells that migrated to the lower side of the membrane were fixed, stained, and counted. g One hundred thousand SW-13, SW-Neo, and SW-DKK3 cells were allowed to invade through Matrigel in modified Boyden chambers for 24 h. Cells that invaded through Matrigel and migrated to the lower side of the membrane were fixed, stained, and counted