Figure 4.

GSK621-mediated AMPK activation decreases the proliferation of mature erythroblasts. (A) GSK621 dose-dependent activation of AMPK in erythroblasts. Western blot analysis of PT172 AMPKα1, PS79 ACC and PS555 ULK1 in primary erythroblasts incubated for 3 h with increasing doses of GSK621. Anti-HSC70 was used as a loading control. (B) GSK621 induces massive cell death from days 5-7. The percentage of dead cells was determined by trypan blue exclusion dye. In the three experiments, the days of culture were grouped at the same stage of differentiation for the vehicle-treated cells (see Methods section). (C) Inhibition of erythroblast proliferation by GSK621. Erythroid cells were incubated in the absence (vehicle) or presence of 20 μM GSK621 from day 0. Cumulative cell number was determined by counting cells with the trypan blue exclusion method at day 0, days 3-4, days 5-7 and days 8-9 in three independent cultures. (D) GSK621 induces an accumulation of cells in the S phase. Erythroid cells were incubated in the absence (vehicle) or presence of 20 μM GSK621 from day 0 to the indicated days. The propidium Iodide incorporation assay was performed. A representative experiment is shown; four independent experiments were performed and the results from day 9 are expressed as the mean ± SD; ns: non-significant, ^*P<0.05. AMPK: AMP-activated protein kinase; ACC: acetyl-CoA-carboxylase; ULK1: Unc-51 like autophagy activating kinase 1; HSC70: heat shock 70kDa protein; d: day.