Figure 4. CENP-A α-amino methylation required for maintaining bipolar spindle.
(a) Schematic of the CENP-A shRNA treatment of parental and CENP-A-eGFP expressing HeLa cells. (b) Rescue with methylation deficient mutants of CENP-A fails to rescue multipolar spindle defects. (c) Quantitation of the multipolar cells. n=3. Error bars indicate s.d. Indicated P values were determined by χ2 test. (d) Western blot showing reduction of CENP-A protein following shRNA treatment. (e) CENP-A knockdown caused cells to develop multipolar spindles. Cells were stained for α-tubulin after CENP-A suppression by shRNA. (f) Quantitation of multipolar cells following CENP-A shRNA treatment. n=3. Error bars indicate s.d. Indicated P values were determined by χ2 test. (g,h) Number of centrioles in the multipolar cells formed by knockdown and replacement with the wild type and mutant CENP-A. The majority of cells have four centrioles, consistent with normal cytokinesis and centriole duplication. n>25 mitotic cells. Scale bars, 10 μm.