Figure 7.

GSK621-mediated AMPK activation leads to autophagy and apoptosis. (A) GSK621-mediated AMPK activation induces autophagy. Erythroid cells were incubated in the absence (vehicle) or presence of 20 μM GSK621 from day 0 to the indicated days of culture (left panel). Chloroquine (10 μM) was added for 4 h before harvesting the cells (right panel). LC3BII was detected by western blot experiments using specific antibodies. Anti-β actin or anti-HSC70 antibodies were used as loading controls. (B) GSK621 provokes the caspase-dependent apoptosis of mature day 8 erythroblasts. Mature erythroblasts were incubated in the absence (vehicle) or presence of 20 μM GSK621 or 20 μM GSK621 and 10 μM QVD for 48 h; cells were labeled with anti-GPA and for annexin V binding and analyzed by flow cytometry. A representative experiment and the percentage of annexin V-positive cells from three experiments are shown. Results are expressed as the mean ± SD. ^*P<0.05, ^**P<0.01. Efficiency of QVD to block caspase activity was determined by western blot using an anti-caspase 3 antibody that specifically detects the cleaved isoform of caspase 3. β-actin was used as a loading control. d: day; CQ: chloroquine; GPA: glycophorin A; QVD: Q-VD-OPh.