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Figure 2


Fig. 2. Rac1 and Rac3 produce opposite effects on N1E-115 morphology. (A) Cells were transfected with pcDNA3/Rac1~HA, pcDNA3/Rac3~FLAG, pSuper/shRac1 or pSuper/shRac3, together with a green fluorescent protein vector (ratio 10:1). Subsequently, cells were serum-starved for 24 hours, fixed and stained with phalloidin to visualize filamentous actin. Bar, 25 µm. (B) Quantification of the changes in morphology as seen in A from at least three independent experiments (50-100 cells counted per experiment). Cells were scored as flat, round or protrusion bearing. P values: protrusions in Rac1 versus control *P=0.2; rounded cells in Rac33 vs control **P=0.014; rounded cells siRac1 vs control ***P=0.038; protrusion outgrowth in siRac3 vs control ****P=0.003. (C) Cells were transfected with pcDNA3.1 empty vector (ev), mouse-specific shRac3 together with ev, or mouse-specific shRac3 combined with human Rac3 cDNA. Protrusion-bearing cells (where protrusion is >1x cell body) were counted and depicted in a bar graph. Note that the phenotype induced by mouse-specific shRac3 is prevented by co-expression of human Rac3. (D) Rac3 is downregulated in differentiated serum-starved N1E-115 cells. Serum-cultured and serum-starved N1E-115 cells were analyzed by RT-PCR for the amount of Rac1 and Rac3 mRNA. (E) Rac3 depletion induces differentiation of N1E-115 cells as shown by various differentiation markers. Parental N1E-115 cells were either transfected with shRac3 (together with eGFP in a ratio of 10:1, to track the transfected cells) and cultured under normal conditions (right panels) or differentiated by 24 hours serum-starvation and 4 hours NGF treatment (left panels). The cells were stained with either anti-neurofilament 200 (NF200) antibody (upper panels) or anti-microtubule-associated protein 2 antibody (lower panels).





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