Figure 5

Role of moesin in HA-induced migration in glioma cells. (A) Western blot analysis. Both the glioma cell lines (U87 and U373) were transfected with siRNA targeting moesin and scrambled siRNA used as a negative control for 48 h followed by Western blotting. Panel represents shows effective downregulation (>70%) of moesin in both U87 and U373 cells in 48 h as compared with no transfection controls (NTC). Cells transfected with scrambled siRNA showed no significant change in the expression of moesin. β-actin was used as a loading control in western blot; (B) Glioma cells (U373 and U87) were transfected with moesin siRNA (200 nM) followed by treatment HA (100 μg/mL,48 h) and analyzed using wound healing assay as described in Materials and Methods. Panel shows number of cells migrating in the wound (i) no treatment controls (NTC); (ii) HA treatment (100 μg/mL, 48 h); (iii) cells transfected with moesin siRNA (200 nM, 48 h); (iv) cells transfected with moesin siRNA followed by treatment with HA (Original magnification X40). (C) Bar graph showing relative percentage of glioma cells (U373 / U87) that migrated into the lower chamber of transwell plates in no treatment controls (NTC), glioma cells treated with HA only, cells transfected with moesin siRNA (i.e. the cells with reduced moesin expression) and glioma cells transfected with siRNA targeting moesin followed by treatment with HA. As shown in the graph, glioma cells showing reduced expression of moesin demonstrated significantly reduced migration in transwell chambers even after treatment with HA (n = 3, *p-value < 0.01). This clearly demonstrates the significance of moesin downstream of HA-CD44 interaction in glioma cells.