Figure 4.

Reggie-1 knockdown impairs process formation in N2a cells. Cells transfected with either GL2 control siRNAs or a mix of reggie-1-specific siRNAs were stimulated with IGF-1, fixed 24 h after stimulation, and stained with phalloidin to visualize F-actin and cell morphology. Untreated and GL2 siRNA-transfected cells produced numerous filopodia (A, B), whereas reggie-1 downregulation (R1 siRNA) led to the formation of large lamellipodia (F). Significantly fewer cells formed neurites after reggie-1 siRNA transfection (E), which efficiently downregulated reggie-1 and reggie-2 expression compared with GL2 siRNA cells in Western blot experiments (G). D, N2a cells were simultaneously transfected with reggie-1 siRNA and the reggie-1 construct (R1–EGFP rescue) without siRNA-binding sites. A large proportion of the cells no longer exhibited the lamellipodia-rich phenotype but had instead many filopodia and neurites (D–F) much as control cells (A, B). Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. Scale bars, 20 μm.