Fig. 6.

Slug knockdown decreases NUMB6-induced DB-7 cell migration and invasion in vitro. NUMB4-GFP, NUMB6-GFP or control vector GFP DB-7 cells were treated with 2.5uM or 5 uM Slug siRNA or control siRNA; NUMB6-GFP DB-7 cell migration was reduced with Slug siRNA treatment assessed in scratch assay and transwell assay. (A) In scratch assay monolayers of NUMB4-GFP, NUMB6-GFP, and GFP DB-7 cells transfected with control siRNA or Slug siRNA were incubated at 37°C until confluence, then wounded with a uniform scratch, washed to remove cell debris, and cultured for indicated times in the completed DMEM-F12 medium. Images of cell cultures were captured at 0 and 24 h after scratching. (B) In transwell assay DB-7 cells overexpressing GFP, NUMB4-GFP, or NUMB6-GFP were treated with control siRNA or Slug siRNA for 48hr. Cell were then trypsinized, counted and 1x10⁴ cells were loaded into chamber inserts containing an 8-μm pore size membrane and migrated toward 10% FBS. The non-migrated cells were removed from the top of the membrane by cotton swap whereas migrated cells were fixed and stained with Hoechst 33342 after 20 h incubation at 37 °C. The migrated cells were counted. (C) Matrigel invasion assay of DB-7 cells overexpressing GFP, NUMB4-GFP, or NUMB6-GFP treated with control siRNA or Slug siRNA for 48hr. Cell were trypsinized, counted and 1x10⁴ cells were plated then in the invasion chamber and incubated at 37 °C for 20 h. The non-migrated cells were removed from the top of the membrane by cotton swap whereas migrated cells were fixed and stained with Hoechst 33342. The migrated cells were counted. Each experiment was performed in triplicate and repeated a minimum of three times. Error bars represent SEM. Asterisks indicate significant differences between two groups.