Figure 1.

AICAR treatment leads to cell cycle arrest by p53/p21 induction, without effects on apoptosis. (A) AICAR activates p53/p21 pathway in R1 mES cells. Cells were treated with 0.5mM AICAR for 1, 6, or 9 h, and then cell lysates were prepared. Total lysates were analyzed by immunoblotting for phospho-ACC, ACC, phospho-STAT3, p53 and p21 expression. Anti-β-tubulin antibodies were used as loading controls. A representative blots of at least three different experiments is shown. (B) AICAR inhibits proliferation of mES cells via p53/p21 pathway. Proliferation of mES cells expressing scrambled shRNA, p53 shRNA or p21 shRNA 1d after AICAR treatment (0.5 mM). 5 × 10⁴ cells were seeded in 6-well plates. After 12h, cells were cultured with or without AICAR (0.5 mM) for 1 day and viable cell number was measured by Tryphan blue exclusion and results displayed as % of numbers of control cells which were expressing corresponding shRNA, but were not treated with AICAR. (C) AICAR does not affect mES cell viability. Cells were treated with or without AICAR (0.5 mM, 24 h), and cell viability was assessed by AnnexinV/7-AAD staining. (D) Knock-down of p53 and p21 expression. mES cells were infected by scrambled shRNA-, p53 shRNA-, or p21 shRNA-expressing lentiviruses. Expression of p53 and p21was analyzed by immunoblotting using the cells treated with or without AICAR for 9h. (E) AICAR induces cell cycle arrest of mES cells. Cells were treated with AICAR (0.5 mM) for 1 day. Cells were subsequently incubated with BrdU for 15 min, fixed, stained with anti-BrdU-FITC Ab and 7-AAD. Bivariate analysis of total DNA content and BrdU incorporation was performed by flow cytometry. Plots are representative of three or more experiments with similar results. Data represent % cell populations residing at each cell cycle stage determined by DNA content and BrdU incorporation, and is expressed as mean ± SD (n=3). * p<0.05, ** p<0.01.