Figure 5. Slug is necessary and sufficient for cyclin D1b–mediated prometastatic and tumorigenic phenotypes.

(A) Control and cyclin D1b cells were treated with a pool of siRNAs directed against the SNAI2 transcript for 72 hours and harvested for RNA (left) and protein (right). Relative levels of SNAI2 and Slug are reported. (B) siRNA-treated cells were cotransfected with GFP and allowed to invade the Boyden chamber matrix toward an androgen-proficient gradient for 24 hours, after which they were fixed and GFP-positive cells counted. Left panel shows representative fields of GFP-positive cells. Original magnification, ×10. (C) LNCaP control, Slug-expressing, and Slug- and cyclin D1b–expressing cells (left) were plated in soft agar and cultured in androgen-proficient conditions for 4 weeks. Colonies greater than 75 μm were counted for colony formation (middle). Right panel shows representative colony growth for each cell line after 4 weeks. Original magnification, ×20 (inset). (D) A polyclonal population of cells expressing Slug was probed by immunofluorescence for DAPI (panel 1), Slug (panel 2) and E-cadherin (panel 3). Merged images are represented in panel 4. Original magnification, ×40. DAPI serves as a nuclear control. Error bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.