Fig 2: Low level inhibition of AurkA does not affect MEFs cell cycle or mES differentiation.(a) 4F-infected MEFs were treated with different concentrations of B6 on day 3 post-infection. Cells were collected 48 h later, subjected to propidium iodide staining and analysed for DNA content by flow cytometry. (b) MLN8237 treatment at 10 nM did not alter the cell cycle of 4F-infected MEFs. The experimental procedure was as in a. (c) Aurora A kinase inhibitors do not inhibit mESC differentiation. Treatment with inhibitor B6 does not block changes in mESC morphology following LIF withdrawal. mESCs were cultured in LIF+ (w/LIF) or LIF-(w/oLIF) medium for 4 days and collected for AP staining. Differentiating cells become AP-negative and show a more scattered morphology. (d) Inhibitor B6 does not affect silencing of self-renewal genes following LIF withdrawal. The experimental procedure was as in c. Cells were collected for RNA extraction and RT–qPCR of the indicated mESC self-renewal genes. Data are mean±s.d. of triplicate samples.

Fig 3: Inhibition of Aurora A kinase by B6 promotes inactivation of GSK3ß.(a) Both mock-infected and 4F-infected (day 3) MEFs were treated with 1 µM B6 for 2 days and then collected for western blotting of AurkA. Actin served as a loading control. (b) MEFs were infected with 4F for 3 days and mock-treated or treated with DMSO or 1 µM B6 for 2 days before isolation of total RNA and RT–qPCR analysis. B6 treatment did not alter induction of AurkA by 4F. Error bar represents variation between two experiments with triplicate wells. (c) 4F-infected MEFs were treated with the indicated doses of B6 inhibitor starting on day 3 post-infection and 2 days later were collected for western blotting analysis. (d) Inhibition of AurkA by MLN8237 also increases AurkA protein level and dose-dependently promotes GSK3ß phosphorylation. The experiment was performed the same as for c. Actin served as the loading control. (e) Expression of a dominant-negative form of AurkA promotes GSK3ß (Ser9) phosphorylation. MEFs were infected with 4F plus expression vectors for red fluorescent protein (RFP), wild-type (wt) murine AurkA or the D274A kinase-dead mutant of human AurkA. Expression of wt AurkA inhibited GSK3ß phosphorylation on Ser9, whereas overexpression of the mutant AurkA D274A enhanced GSK3ß phosphorylation. Exposure time was almost doubled for 4F-infected samples. (f) MEFs were transfected with 50 nM AurkA and control siRNAs for 2 days. Cells were then collected for western blotting of AurkA, total and phosphorylated GSK3ß (Ser9), and actin as a loading control. (g) MEFs were infected with pMX-RFP (control) or pMX-GSK3ß virus for 4 days and then collected for RT–qPCR analysis. Error bar represents variation of duplicate wells. (h) Overexpression of GSK3ß blocked the effect of B6 on reprogramming. pMX-Gsk3ß was transduced into MEFs together with 4F viruses. B6 (1 µM) was added at day 3 post-transduction and GFP+ colonies were quantified on day 12 post-transduction. Data are mean±s.d. of three independent wells. *P<0.05 (Student's t-test).