Figure 3. AMPK knockdown inhibits MMC-induced activation of FANCD2.
A. MMC-induced FANCD2 monoubiquitination is reduced in AMPKα siRNA-transfected U2OS cells. U2OS cells were transfected with siRNA specific to AMPKα1 (siAMPK#8) or control siRNA (siControl). After 64 h, MMC (25 ng/mL) was added and the cells incubated for 8 h. Monoubiquitinated FANCD2 (FANCD2-L) and unmodified FANCD2 (FANCD2-S) were visualized by immunoblotting (top panel). The ratios (L/S) of band intensities of FANCD2-L and FANCD2-S are shown below the panel. Knockdown efficiency was assessed by immunoblotting with anti-AMPKα (middle panel) and anti-β-actin (bottom panel) antibodies. B. Formation of FANCD2 nuclear foci is inhibited in AMPKα siRNA-transfected cells. U2OS cells grown on coverslips were transfected with siAMPK#8 and then treated with MMC. FANCD2 nuclear foci (green) were visualized by immunofluorescence staining and confocal microscopy. Cells were counterstained with DAPI to stain the nuclei (blue). Representative images of MMC-treated samples are shown in the left. The number of foci per cell was counted and plotted for ≥ 13 cells (right panel). The values represent the mean ± SEM. (Student's t-test, *, P < 0.05). C. Sensitization of U2OS cells following AMPKα knockdown. U2OS cells were transfected with siControl or siAMPK#8 and then treated with MMC (serial two-fold dilutions from 400 ng/mL) in triplicate for 6 days. Cell viability was measured using MTT assays, and the percent survival was calculated for comparison with untreated cells. A representative graph from three independent experiments is shown. The values represent the mean ± SD. (Student's t-test, **, P < 0.01; ***, P < 0.001).