GSK3 Regulates Mitotic Chromosomal Alignment through CRMP4
Figure 10
siRNA-mediated knockdown of CRMP4 alters spindle morphology.
(A) HeLa cells were transfected with either control or CRMP4 siRNA, and for rescue experiments were co-transfected with either L-CRMP4-WT-V5 or L-CRMP4-AAA-V5. In some experiments, HeLa cells were treated with SB-216763 (10 um) for 90 mins. HeLa cells were permeabilized with a cytosolic extraction buffer prior to fixation with 4% PFA and immunofluorescence detection of α-tubulin and DNA. Bar, 10 um. (B) Bar graph plotting pole to pole distance. HeLa cells transfected with CRMP4 siRNA had a shorter pole to pole distance (n = 3, **p = 0.0059, Student's t test compared to control siRNA). Co-transfection with a L-CRMP4-WT-V5 construct rescued the shorter pole to pole distance phenotype (n = 3, **p = 0.0069, Student's t test compared to CRMP4 siRNA) while a L-CRMP4-AAA-V5 construct did not rescue (n = 3, ns = non significant p>0.05, Student's t test compared to CRMP4 siRNA). HeLa cells treated with SB-216763 (10 uM) for 90 mins resulted in a shorter pole to pole distance (n = 3, ***p<0.0001, Student's t test compared to control siRNA; n = 3, *p = 0.0322, Student's t test compared to CRMP4 siRNA). (C) Bar graph plotting spindle width. HeLa cells transfected with either control siRNA, CRMP4 siRNA or treated with SB-216763 did not show any differences in spindle width (n = 3, ns = non significant p>0.05, Student's t test compared to control siRNA). n = 3 refers to 3 independent experiments where at least 20 cells were analyzed per experiment.