Figure 6. Active AMPK associates with the mitotic apparatus. (A) Untreated A549 cells were fixed and stained with Hoechst (blue) and an antibody that detects phosphorylated AMPKα on its Thr172 residue (green, 1:00 dilution, Cell Signaling). The cells were imaged at 40× progressing through each phase of mitosis. A representative image for each phase is shown. (B) A549 cells were left untreated or exposed to a single dose of 8Gy IR and imaged one hour later. IR enhanced nuclear and cytoplasmic AMPK phosphorylation (green) in mitotic (arrow) and interphase cells. (C) AMPK activity in A549 cells was inhibited using either the chemical agent compound C (Comp C, 1 μM, Calbiochem) or molecular knockdown of AMPKα with siRNA (Qiagen). AMPK phoshporylation (green) was present in untreated dividing cells, but blocked in cells treated with Comp C or AMPKα siRNA. Phosphorylated histone H3 (Ser10, in red 1:100 dilution, Cell Signaling), which occurs during chromosome condensation during mitosis, was used here are a marker of mitotic progression. Phospho-Ser10-H3 signal and mitosis were detected despite inhibition of AMPK. (D) Mouse embryonic fibroblasts (MEF) that are wild-type or lack AMPKα expression (AMPKα1/2^−/−) were fixed and stained with Hoechst (blue) and an antibody that detects phosphorylated AMPKα on its Thr172 residue (red). Dividing and non-dividing cells were imaged at 40× and a representative section of cells are shown.