(A) Phosphorylation of endogenous AMPK, PPP1R12C, and PAK2 in U2OS cells synchronized by thymidine followed by monastrol block and collected at different time points after release from the block (blue bar). Cell lysates were analyzed by western blot.
(B) AMPK FRET signal is high when cells enter mitosis and starts to decrease around the time the cells initiated cytokinesis in U2OS cells expressing AMPKAR, a fluorescent reporter for AMPK activity. AMPK activity was measured at 10 min intervals. Red: increased AMPK activity.
(C) U2OS cells were synchronized by thymidine followed by monastrol block and chemicals were added 1.5 hours after the release from the monastrol block (blue bar) and replating of mitotic cells. Cells were fixed 4.5 hours after the addition of chemicals. The number of multinucleated cells was normalized to the number observed in non-treated control cells for each experiment. Means +/− SEM of 3 independent experiments are shown, except for the LY-294002 condition, which represents 2 independent experiments. *p<0.05 by one-way ANOVA analysis. ns: not statistically significant.
(D) U2OS cells expressing WT or S452A PPP1R12C were synchronized by thymidine block. Cells were fixed at the indicated time post-release from the block (blue bar). Percent of multinucleated cells in each condition are shown. Means +/− SEM of 3 independent staining for a synchronization experiment. *p<0.05, **p<0.01, ***p<0.001 by 2-way ANOVA, with Bonferroni post-hoc tests.
(E) AMPK directly phosphorylates a number of substrates involved in coordinating different aspects of mitosis.