Effects of 2-DG on LEF2 cells. A) Wild-type RK3E and Tsc2-/- LEF2 cells were grown in 10%FBS DMEM with 2 mM glucose. Left: Phase contrast photomicrographs of cells after 2 days. Magnification 10×. Right: Immunoblot of cell lysates showing expression of cleaved PARP and α-tubulin at indicated times. Effects of glucose concentration and 2-DG (4 mM) for 6 hours on B) cell viability as determined by Annexin V/PI flow cytometric analyses, C) cellular ATP levels (Luminescence assay, PerkinElmer), error bars represent SEM. *, p < 0.05, and D) AMPK phosphorylation and p62 expression by Western blotting. The relative expression of these proteins were analyzed by densitometry, and the numbers under the blots indicate the ratio of band intensities (Image J) between p-AMPK:total AMPK and p62:α-tubulin. The densitometry results are also shown in graphical form on the right. Arrows indicate the most significant changes.