Fig 2: Up-regulation of MLC2 phosphorylation. A, Immunoblotting of the Vil2+/+ and Vil2kd/kd neurons (2 DIV) using antibody recognizing phospho-MLC2 (Ser19, top), MLC2 (middle) and GAPDH (bottom), respectively. Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. B, The ratio of phosphorylated MLC2 to total MLC2 in the lysate of the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons was shown. C, Immunoblotting of the Vil2+/+ and Vil2kd/kd neurons (2 DIV) using antibody recognizing phospho-ERM (top), pan-ERM (middle) and GAPDH (bottom), respectively. Representative blotting patterns were shown. 8 µg of cell lysate was applied onto each lane. D, The ratios of phosphorylated ezrin, radixin and moesin to each total protein in the lysate of the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons were shown. Each experiment was performed in triplicate. **p<0.01 , Student's t test. Data represent mean ± SE.

Fig 3: Neuritogenesis is impaired by ezrin knockdown. A, B, The Vil2+/+ (A) and Vil2kd/kd (B) neurons were fixed at 2 DIV and stained with an anti-neuronal class III ß-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. C, Stacked bar graph showing stage progression in the Vil2+/+ (n = 153) and Vil2kd/kd (n = 162) neurons. Stage of cells were defined by the length of the longest neurite as reported previously [19]. D-F, Quantitation of number (D) and length (E) of neurites, and length of axon (F) in the Vil2+/+ (gray columns, n = 50) and Vil2kd/kd (green columns, n = 50) neurons. Three independent experiments were performed. *p<0.05, **p<0.01, ***p<0.001, Student's t test. Data represent mean ± SE.

Fig 4: Y-27632 rescues neuritogenesis. A-D, The Vil2+/+ and Vil2kd/kd neurons treated with DMSO (A,C) or 40 µM Y-27632 (24 h, B,D) were fixed at 2 DIV and stained with an anti-neuronal class III ß-tubulin antibody (green) and rhodamine phalloidin (red). Scale bars, 50 µm. E-G, The number (E) and length (F) of neurites, and length of axon (G) were quantified in the Vil2+/+ and Vil2kd/kd neurons treated with DMSO (white columns, n = 30) or 40 µM Y-27632 (black columns, n = 30). Three independent experiments were performed. *p<0.05, **p<0.01, ***p<0.001 (DMSO-treated vs. Y-27632-treated), ###p<0.001 (DMSO-treated Vil2+/+ vs. DMSO-treated Vil2kd/kd), Student's t test. Data represent mean ± SE.

Fig 5: Increased RhoA activity in the Vil2kd/kd neurons. A-C, The amounts of active and total RhoA (A), Rac1 (B) and Cdc42 (C) from cell lysates of the Vil2+/+ and Vil2kd/kd neurons (2 DIV). Representative patterns were presented. D-F, The ratios of active RhoA (D), Rac1 (E) and Cdc42 (F) to total amount of proteins were compared between the Vil2+/+ (white columns) and Vil2kd/kd (black columns) neurons. Each experiment was performed in triplicate. *p<0.05 , Student's t test. Data represent mean ± SE.