Fig. 3

TPX2 is the downstream effector of COASY leading to multinucleation and extended mitosis. a COASY knockdown increased three acetylated peptides (K75, K476, and K582) of TPX2 as determined by acetylome analysis. b COASY knockdown increased TPX2 protein level in A549 cells. c COASY knockdown increased the TPX2 acetylation in A549 cells. A549 cells enriched in early mitosis by thymidine-nocodazole block were transfected with control or COASY siRNA and probed with indicated antibodies The TPX2 in the cell lysates were then immunoprecipitated and blotted for pan-acetylated lysine antibody. d COASY knockdown decreased TPX2 ubiquitination in HEK-293T cells. HEK-293T cells were transfected with COASY siRNA and TPX2 cDNA. After MG132 treatment, TPX2 were immunoprecipitated and blotted for pan-ubiquitination antibody. e TPX2 protein expression level and acetylation on TPX2 regulated the percentage of COASY-dependent multinucleation. TPX2 knockdown rescued the multinucleation induced by COASY knockdown. Reintroducing wild type (WT) or acetylation-mimetic mutant (3Q), but not acetylation-deficient (3R), of siRNA-resistant TPX2 cDNA recapitulated multinucleation phenotype. Multinucleation was determined as described in the legend to Fig. 1. f COASY knockdown triggered increased Aurora A Thr 288 phosphorylation and extended mitosis by the elevation of TPX2. A549 cells were synchronized by thymidine-nocodazole block and released in fresh media for the indicated time (in min). COASY knockdown increased TPX2 expression and delayed its decline during from 40 min (control) to >160 min. COASY knockdown also delayed the inactivation of Aurora A and degradation of cyclin B1, indicating extended mitosis. The extended mitosis caused by COASY knockdown can be abolished by simultaneous TPX2 knockdown. Bars show standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test, n = 3 independent repeats