FIGURE 3.

Knockdown of CRMP2 diminishes spindle pole-to-cortex distance and astral MTs. A, representative confocal images of RO-3306-synchronized cells released for 40 min before fixation. Panels show control siRNA- or CRMP2 siRNA-treated OLDN-93 cells stained with anti-γ tubulin, phalloidin, Hoechst dye, and anti-CRMP2 antibodies. The inset represents an enlarged image of the γ-tubulin-rich spot and the cortical actin. Scale bar = 5 μm. Cells were imaged using a 60× oil lens of a confocal microscope. B, representative deconvoluted confocal images of RO-3306-synchronized, control siRNA-, or CRMP2 siRNA-treated OLDN-93 cells fixed at 40 min after release. The cells were stained with anti-α-tubulin, Hoechst dye, and anti-CRMP2 antibodies. The inset represents an enlarged image of the region pointed out by the white arrow. Cells were imaged using a 100× oil lens of a confocal microscope. Deconvolution was performed, and Z projections are shown. C, left, a schematic diagram illustrating the spindle pole-to-cortex distance measurement between the γ-tubulin spot and the cortical actin. Right, a graph representing the average spindle pole-to-cortex distance for control and CRMP2 siRNA-treated cells. Cells with membrane protrusions at the cortex were excluded during the quantitation. Error bars represent S.D. between three independent experiments with the number of cells (n) counted for each condition labeled below the graph. (*, p < 0.05, Student's t tests when compared with control). Ctrl SiRNA, control siRNA.