Figure 2. RasGRP3 regulates the migration and invasion of glioma cells.

Expression of exogenous RasGRP3 in stable clones of U87 is shown in a Western blot compared with control vector cells (CV) using anti-FLAG antibody (A). Cell migration was assayed using a transwell chamber containing polycarbonate membrane inserts of 8-μm pore size (Corning, Costar) with fibronectin coating. U87 cells overexpressing RasGRP3 (3 × 10⁴ cells) in serum-free DMEM were added to the upper chamber. The bottom chamber was filled with 600 μl of serum-containing medium, and the assembly was incubated at 37ºC for 3–4 hrs to allow cell migration. The cells were removed from the top of the membrane and the migratory cells were stained and quantified. Values shown are the mean ± SE of triplicate experiments (B). A172 cells were transfected with control or RasGRP3 siRNA pools and the expression of the RasGRP3 protein was determined by Western blot analysis (C). Cell migration was determined as described in Methods (D). The results represent the means ± SE of three independent experiments (P < 0.01). A172 cells transfected with control siRNA or RasGRP3 siRNAs were plated onto BD invasion chambers (8-μm pore size with polycarbonate membranes). Matrigel invasion assays of A172 cells transfected with control or RasGRP3 siRNAs were performed as described in Methods (E). Representative images are shown. The results were quantitated and the graph presents the means ± SE of three independent experiments (P < 0.01). Matrigel invasion assay was determined also for the LNZ308 glioma cells transfected with either control or RasGRP3 siRNAs (F). In the confrontation assay, spheroids of U87 cells overexpressing RasGRP3 or a control vector labeled with Green Cell Tracker were confronted with astrocytic spheroids labeled with Red Cell Tracker and cell invasion was determined after 3 days (G). (H–I) The effect of RasGRP3 silencing was also examined on the migration (transwell assay) and invasion (matrigel invasion assay) in the HF2354 (H) and HF2607 (I) GSCs. For these experiments, GSC neurospheres that were silenced for RasGRP3 for 3 days were disaggregated and analyzed for the migration and invasion assays as described for the glioma cell lines. The results represent three different experiments, each consisted of 6 cultures that gave similar results. *p < 0.005