Aurora A inhibition rescues the PPP6C depletion phenotype. (A) HeLa cells transfected for 48 h with control and PPP6C si08 duplexes were treated with 10 or 20 nM MLN8237 or a solvent control for 15 min before lysis in phosphatase inhibitor containing buffer or fixation. Total lysates were analyzed by Western blotting. The red and black lines indicate the phosphorylated and nonphosphorylated forms of Aurora A. Fixed cells were stained using DAPI to detect DNA and antibodies to α-tubulin and Aurora A pT288. The intensity of pT288 staining was integrated using ImageJ over the spindle region defined by TPX2 staining and is plotted in the bar graph (n = 4). Arrowheads indicate micronuclei. Bar, 5 µm. (B) HeLa cells transfected for 48 h with control and PPP6C si08 duplexes were treated with 10 nM MLN8237 or a solvent control for 24 h before fixation and staining with DAPI to detect DNA and CREST to mark centromeres. Micronucleation is quantitated in the graph (n = 3). (C) HeLa cells were transfected for 48 h with control, PPP6C si08, and TPX2 duplexes alone or in combination before fixation. Cells were stained using DAPI to detect DNA and CREST to mark centromeres. The graph shows the percentages of normal and micronucleated cells for the various conditions (n = 3). (B and C) Arrowheads indicate CREST staining outside the main nucleus defined by the DAPI staining. Bars, 10 µm. Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean.