Fig. 2

Prolonged treatment with AKTi induces FOXO3a acetylation and BRD4 association. a BT474 and T47D cells were treated with 1 μM MK2206 for 6 h, the cellular localization of FOXO3a (green) and SirT6 (red) were analyzed by immunofluorescent staining. Nuclei stained with DAPI (blue). Scale bar, 50 μM. b Schematic diagram showing the domain structure of FOXO family members. Sequence alignment indicates the highly conserved GK-X-GK motif among K5/8 of histone H4, K73/76 of Twist, K242/245 of FOXO3a, in which the lysine residues have been reported to be acetylated in vivo. c BT474 and T47D cells were treated with AKTi for 4 day, cell extracts were subjected to immunoprecipitation (IP) with FOXO3a antibody, the acetylation of endogenous FOXO3a and its interaction with BRD4 were analyzed by western blot using pan acetylated-lysine and BRD4 antibodies. d BT474 cells were treated with 1 μM MK2206 for various time intervals. Cell extracts were prepared for IP with FOXO3a antibody, the acetylation of endogenous FOXO3a and bound BRD4 were analyzed by western blotting. e BT474 cells were treated with 1 μM MK2206 for 4 days. Cell extracts were prepared for IP with FOXO3a antibody in a buffer containing either 2 μM JQ1 or MS417. Western blottings were used to detect FOXO3a acetylation and its interaction with BRD4. f BT474 cells were treated with 2 μM of TSA and 4 mM of Nicotinamide overnight. Cell extracts were prepared and FOXO3a were IP in a buffer containing either JQ1 or MS417 as described above. FOXO3a acetylation and its interaction with BRD4 were analyzed by western blotting