Fig. 4.

Deficient mitotic spindle assembly in Samp1-depleted cells. HeLa cells treated with siRNA and synchronised in metaphase were immunostained using antibodies specific for β-tubulin and pericentrin, and DNA was stained with DRAQ5. (A) Representative images of β-tubulin staining of metaphase spindles in cells treated with control, scrambled siRNA (siScrambled) or siRNA against Samp1 (siSamp1). Scale bar: 10 µm. (B) Quantification of β-tubulin immunofluorescence intensities from siScrambled (grey), siSamp1a oligo3 (white) and Samp1a–YFP+siSamp1a oligo3 (striped) metaphase spindles (n=150 cells, ****P<0.0001, unpaired two-tailed Student's t-test). AU, arbitrary units. (C) Quantification of the metaphase plate area, measured as z-stack maximum projections of HeLa cells treated with siScrambled (grey) or siSamp1 oligo1 (white) (****P<0.0001, n=50 cells, four replicates, unpaired two-tailed Student's t-test). (D) Spindle length. Representative images showing pericentrin and Samp1 immunostaining in siRNA-treated cells in metaphase. The spindle pole-to-pole distances were measured. Scale bar: 10 µm. (E) Quantification of spindle pole-to-pole distance as shown in D, which was significantly increased in Samp1-depleted cells (white) compared to control cells (grey) (****P<0.0001, n=50 cells, three replicates, unpaired two-tailed Student's t-test). (F) Quantification of metaphase spindle pole-to-pole distance in cells treated with yet another siRNA oligonucleotide pair specific for Samp1a showed a significant increase in spindle length between siSamp1a-treated cells (white) compared to siScrambled (grey). Co-transfection of HeLa cells with cDNA encoding siRNA resistant Samp1a–YFP and siSamp1a (striped) rescued the increased spindle length phenotype (*P<0.05, **P<0.01; NS, not significant; n=150 cells, three replicates, unpaired two-tailed Student's t-test). Box plots are presented as described in the Materials and Methods.