Induction of Ulk1 expression correlates with onset of autophagy and loss of mitochondria during terminal erythroid maturation. Erythroid maturation was examined using the FVA culture system described previously.40 (A) May-Grünwald and benzidine (inset) stains of splenic erythroblasts cultured in the presence of erythropoietin (EPO) for 1 hour (left panel) and 48 hours (center panel). The right panel shows the relative distribution of cells at various stages of erythroid maturation after 1, 24, 36, and 48 hours in culture from a representative experiment. Differentials were performed on more than 300 cells per time point. PE indicates proerythroblast; BE, basophilic erythroblast; PCE, polychromatophilic erythroblast; OCE, orthochromatic erythroblast; and retic, reticulocyte. (B) Quantitative RT-PCR (TaqMan, Applied Biosystems) analysis was performed in triplicate using RNA isolated from cells after 1, 24, 36, and 48 hours in culture. Levels of ulk1 and ulk2 mRNA were normalized to that of 18S RNA. The expression of ulk1 and ulk2 relative to levels in control 3T3 cells is shown in the graph. Error bars represent standard deviation. (C) Northern blot analysis of cells after 1, 24, 36, and 48 hours in culture using an ulk1 cDNA probe (bottom panel). The top panel shows levels of ribosomal RNA on an ethidium bromide–stained agarose gel. (D) Western blot analysis of cells after 1, 24, 36, and 48 hours in culture. (E) Quantitative RT-PCR (TaqMan) analysis was performed in triplicate using RNA isolated from cells after 1, 24, 36, and 48 hours in culture. Levels of lc3 RNA levels were normalized to 18S RNA. The graph shows lc3 RNA levels relative to the 1-hour time point. Error bars represent standard deviation. (F) Representative electron micrographs of autophagosomes containing mitochondria (highlighted by ↗) in a reticulocyte.
