Fig. 5. Knockdown of Vangl2 in vitro perturbs MT organization in Sertoli cells through changes in MT polymerization activity.

a Knockdown of Vangl2 by RNAi using specific Vangl2 siRNA duplexes (Vangl2 RNAi) vs. the non-targeting control siRNA duplexes (Ctrl RNAi) was assessed by immunoblotting wherein the expression of Vangl2 was reduced by ~70%, but also reducing the expression of detyrosinated α-tubulin by ~40% (note: detyrosinated α-tubulin is known to confer MT stabilization, making MT less dynamic). However, the expression of EB1, a +TIP protein known to stabilize MTs, did not alter, with ß-tubulin and ß-actin served as the protein loading control. Some data shown in the left panel were summarized in the bar graph and shown on the right. Each bar is a mean ± SD of n = 3 independent experiments. **P<0.01 by Student’s t-test. b MT spin-down assay was performed using lysates of Sertoli cells transfected with either non-targeting negative control or Vangl2 siRNA duplexes to assess the relative level of polymerized MTs in Sertoli cells following Vangl2 knockdown. Sertoli cells treated with CaCl₂ (4 mM) and Taxol (30 µM) served as the corresponding negative and positive control, in which CaCl₂ is known to promote MT depolymerization whereas Taxol is a MT stabilizing reagent. A considerable decline in the β-tubulin (note: α- and ß-tubulin serve as the building blocks of MT) level following Vangl2 knockdown in the pellet illustrates that Vangl2 is involved in promoting MT polymerization and/or MT stabilization. The histogram below summarizes results of immunoblot analysis such as those shown above with each bar represents a mean ± SD of n = 3 independent experiments using different batches of Sertoli cells. **P<0.01 by Student’s t-test compared to control. c IF analysis was performed using Sertoli cells transfected with either non-targeting control or Vangl2 siRNA duplexes wherein considerable changes in MT organization were detected in Vangl2 silenced cells by α-tubulin staining. For instance, MTs no longer stretched across the entire Sertoli cell cytosol but retracted from cell periphery, moving closer to encircle the cell nucleus. A considerable down-regulation of the fluorescent intensity of detyrosinated a-tubulin following Vangl2 knockdown was observed, consistent with findings shown in (a). Successful transfection was indicated by DY-547-siGLO (red fluorescence; Dharmacon-GE Healthcare) which was used for co-transfection with siRNA duplexes. Scale bar, 25 µm, which applies to other micrographs. d Changes in MT organization in Sertoli cells following Vangl2 knockdown was quantified by comparing the longest distance of MT visualized by α-tubulin staining from the cell periphery to the nucleus (L_l) with the shortest one (L_s) in 70 randomly selected cells from an experiment. Scale bar=20 µm, which applies to other micrographs. A statistically significant difference in the ratio from the Vangl2 knockdown cells vs. control cells was noted in the histogram shown on the right panel. Each bar is a mean ± SD of n = 3 experiments using different batches of Sertoli cells. **P<0.01 by Student’s t-test