Fig. 2

RNAi silencing of DKK3 in ACC cell lines and effects on cell behavior. a Western immunoblot detection of endogenous DKK3 in SW-13 (left) and NCI-H295R (right). b and c, Relative expression of DKK3 as determined by qRT-PCR in siRNA-treated SW-13 (2B) and NCI-H295R (2C) cells, normalized to expression in cells treated with scrambled siRNA for 24 h. d Western immunoblot detection of DKK3 in SW-13 cells treated with control (1), scrambled negative siRNA (2), 10 (3), 20 (4), and 40 nM (5) DKK3 siRNAs for 24 h followed by protein extraction 48 h post-transfection. e-h, NCI-H295R (e and f) or SW-13 (g and h) cells treated with Lipofectamine (Lipo), scrambled negative siRNA (S-ive), or DKK3 siRNA (DKK3) for 24 h, allowed to grow in clonogenic growth conditions (e and g), or allowed to migrate through modified Boyden chambers through growth factor concentration gradient for 12 (f) or 4 (h) hours. Clones with 12 ± 2 cells were fixed, stained, and counted with light microscope (e and g); cells that migrated to lower side of modified Boyden chamber membranes were fixed, stained, and counted (f and h)