Page 64 - 2019_05-HaematologicaMondo-web

M. Ladli et al.
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Figure 1. Expression of AMPK isoforms and AMPK activation along terminal erythroid differentiation. (A) Representative experiment of one ex vivo culture of ery- throblasts derived from CD34+ cells. Cells were analyzed on days 2, 4, 6, 8, 10 and 12 after CD36+ selection. Expression of cell surface markers GPA, band 3 and a4β1 integrin was studied by flow cytometry along terminal erythroid differentiation. The percentage of hemoglobinized cells was determined by benzidine staining. A minimum of 200 cells were counted and the percentage of blue-stained cells among the total cell count was determined. Cell morphology was examined following staining with May-Grünwald-Giemsa; the percentage of each cell population was determined. (B) AMPK isoforms during erythropoiesis were determined by western blot. Protein extracts from human primary erythroblasts from day 2 to day 12 of culture were analyzed by western blot using anti-a1, -a2, -β1, and -γ1 antibodies. Anti-β-actin was used as a loading control and mouse liver protein extracts were used as a positive control for the expression of AMPKa2 (Ctrl). The a1, a2, and γ1 isoforms were analyzed on the same blot, the β1 isoform on a different one. (C) AMPK activation during erythroid differentiation. Anti-pT172 AMPK, anti-p S79 ACC, anti AMPKa1 and LKB1 were used. Anti-β-actin or anti-HSC70 antibodies were used as loading controls. The upper panel shows a representative experiment of three independent ones. Quantification of western blots and determination of the ratio between pAMPK/AMPK, pACC/AMPK and LKB1/AMPKa1 at the indicated days are presented as the mean of three independent experiments ± SD; ns: non-significant, *P<0.05 (lower panel). d: day; GPA: glycophorin A; AMPK: AMP-activated protein kinase; ACC: acetyl-CoA-carboxylase; LKB1: liver kinase B1; HSC70: heat shock 70kDa protein; E: erythroblasts.
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haematologica | 2019; 104(5)