Figure 3

Phenotypic traits of siRNA mediated depletion of ASPM in U2OS cells. A. Reduction in spindle pole associated ASPM (using the anti-ASPM 216-1 antibody), 72 hrs post transfection with ASP1 and ASP2 siRNAs relative to GL3 control dsRNA Scale bar = 10 μm. B. ASPM (216-1 antibody) expression in interphase cells 72 hrs post GL3, ASP1 and ASP2 transfection. Scale Bar = 10 μm. C. Western Blot using rabbit polyclonal anti-ASPM 217-2 antibody, showing ASPM expression in lysates from GL3, ASP1 and ASP2 treated U2OS cells. A β-actin loading control is shown. 25 μg protein for each sample was loaded. D. Celltitre Blue cell viability assay was carried out 72 hours post siRNA treatment, showing a reduction in cell viability in ASP1 and ASP2 siRNA transfected cell cultures. E. Flow cytometric analysis of DNA content and cyclin B1 status 48 and 72 hours post ASPM siRNA treatment, demonstrating a decrease in the number of cells in G2/M and an increase in the number of tetraploid G1 cells. F. Analysis of immunofluorescently labelled cells treated for 48 and 72 h with ASP1 and ASP2 siRNAs. A decrease in mitotic index and an increase in the number of multinucleate cells and cells with centrosome numbers greater than 2 are seen following ASPM knockdown. A decrease in the number of cells displaying morphological hallmarks of cytokinesis, such as the presence of a midbody, is also seen. These observations are consistent with a failure to complete cytokinesis.