Fig. 6: Inhibition of Aurora A abrogates resistance to mitotic stress by YAP1 destabilization.

A Immunoblotting for phospho-Aurora A (pAURKA) after treatment with the indicated dose of MLN8237 (MLN) in P1 or P4 hESCs. Vinculin was used as the loading control. B Relative mRNA levels in P4 hESCs after 24 h of treatment with 0.5 μM MLN (n = 4 independent experiments; mean ± SEM, two-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001). C Flow cytometry analysis of DNA contents. The sub-G1 population (red arrow) is presented after 24 h of treatment with nocodazole (Noc: 50 ng/ml) with or without 0.5 μM MLN (left). Graphical presentation of each phase of the cell cycle (right). D Percentages of live cells quantified by flow cytometry with Annexin V/7-AAD staining. After 4 h of pretreatment with MLN, the cells were treated with 50 ng/ml nocodazole with or without 0.5 µM MLN for 24 h (n = 6 independent experiments; mean ± SEM, two-way ANOVA, ****p < 0.0001). E Immunoblotting for YAP1 in iTPX2-hESCs. After 24 h of treatment with 0.1 μg/ml Dox, 0.5 μM MLN was added for 24 h of treatment with or without Dox. β-Actin was used as an equal loading control. F Immunoblotting for YAP1 in iTPX2-hESCs. YAP1 protein levels after 6 h after treatment with CHX (induction with 0.1 μg/ml TPX2 for 24 h prior to the experiment and then pretreatment with 0.5 μM MLN occurred 1 h prior to CHX treatment). The normalized YAP1 protein band intensity is shown (n = 3 independent experiments; mean ± SEM, two-way ANOVA, **p < 0.01). G Immunoblotting for YAP1 or BCL-xL in iTPX2-hESCs 24 h after treatment with 0.5 µM MLN. H The relative of GFP-positive or GFP-negative live iTPX2-hESC populations (n = 4 independent experiments; mean ± SEM, two-way ANOVA. ****p < 0.000).