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Role of AMPK in human erythropoiesis
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Figure 2. AMPKa1 knockdown reduces erythroblast proliferation without affecting erythroblast maturation. (A) Efficiency of AMPKa1 knockdown: Erythroblasts were infected twice, on days 1 and 4 after CD36 cell sorting, with either a lentivirus coding a scrambled shRNA (sh Ctrl) or an AMPKa1 shRNA (shAMPK a1). The efficiency of AMPKa1 knockdown in human primary erythroblasts was determined by anti-a1 AMPK, anti-PT172 AMPK, anti-PS79 ACC and anti-PS555 ULK1 western blots on day 8 or day 10 with the same results. A representative experiment on day 8 is presented. Western blot quantification was performed with anti-β−actin or anti-HSC70 antibodies; values are the mean of three independent experiments ± SD; ns: non-significant, *P<0.05. (B) Effect of shAMPKa1 on erythroid cell matu- ration. Cell composition for each culture at days 6, 8 and 10. The mean composition ± SD was calculated based on erythroblast morphology determined by May- Grünwald-Giemsa staining in three independent experiments. (C) Absence of AMPKa1 does not modify the percentage of hemoglobinized cells. The percentage of hemoglobinized cells was determined by benzidine staining at the indicated days of culture and corresponding stage of differentiation determined as in Figure 2A. (D) Proliferation of shCtrl versus shAMPKa1 erythroblasts. Cumulative cell number was determined by counting cells with the trypan blue exclusion method at day 6 (basophilic erythroblasts), day 8 (polychromatic erythroblasts) and day 10 (orthochromatic erythroblasts) of culture. Results are expressed as the mean ± SD of three independent experiments; ns: non-significant, *P<0.05. (E) Cell death in shAMPKa1 versus shCtrl cells. The proportion of dead cells was determined by trypan blue exclusion dye at days 6, 8 and 10 of culture. Results are expressed as the mean ± SD of three independent experiments; ns: non-significant, *P<0.05. d: day; GPA: glycophorin A; AMPK: AMP-activated protein kinase; ACC: acetyl-CoA-carboxylase; ULK1: Unc-51 like autophagy activating kinase 1; HSC70: heat shock 70kDa protein; Pro-E: pro-erythroblasts; Baso-E: basophilic erythroblasts; Poly-E: polychromatic erythroblasts; Ortho-E: orthochromatic erythroblasts.
haematologica | 2019; 104(5)
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