Figure 1.
Figure 1. AKT complements EpoR signaling in inducing erythroid differentiation in wild-type and JAK2-/- fetal liver progenitor cells. TER 119- E14 wild-type (A) or JAK2-/- (C) fetal liver cells were transduced with a bicistronic retroviral vector (MIG) or MIG containing the indicated inserts and cultured in the presence or absence of Epo (2 U/mL) on RetroNectin. Thirty-six hours later, live GFP-positive cells from wild-type–(A) or JAK2-/--transduced cells (C) were assayed for CFU-E–generated colony content as determined by diaminobenzidine staining of hemoglobin. Graphs are from duplicates of 4 (A) and 3 (C) independent experiments. The average of the absolute number of CFU-E–derived colonies formed from vector control–transduced cells in the presence of Epo was 3160 ± 64 (n = 4) per 105 wild-type TER 119- cells and 1248 ± 263 per 105 JAK2-transduced JAK2-/-- (n = 3) plated cells. (B) Western blot analysis of transduced NIH 3T3 cells with 1:10 dilution of the retroviral supernatant. The upper band in the activated AKT*-transduced cells corresponds to the Flag-tagged myristylated AKT. (D) Real-time PCR analysis of the β-globin gene in live GFP-positive cells from panel A. Representative graph from duplicate analysis of 2 independent experiments. (E) Real-time PCR analysis of kinetics of up-regulation of β-globin gene expression in TER 119- E14 wild-type fetal liver–derived cells transduced with MIG-AKT* (results expressed as relative to time zero are from total cells). (F) JAK2-/- fetal liver–transduced cells with MIG, MIG-JAK2, or with a constitutively active AKT (MIG-AKT*) and cultured in the presence of Epo (MIG-JAK2 cells) or in the absence of Epo (vector control and MIG-AKT* cells) were analyzed for erythroid-cell differentiation 36 hours later (note TER 119+ cells within transduced GFP-positive cells). (G) Representative field of Wright-Giemsa staining of live GFP-positive transduced JAK2-/--derived cells (magnification, × 1000). Arrows show (i) polychromatophilic erythroblast and (ii) polychromatophilic erythroblast and normoblast. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Garden City, NJ) and a 100 ×/0.3 numeric aperture oil immersion lens. Images were captured using an RT Slider SPOT 2.3.1 camera and SPOT Advanced software (both from Diagnostic Instruments, Sterling Heights, MI).

AKT complements EpoR signaling in inducing erythroid differentiation in wild-type and JAK2-/- fetal liver progenitor cells. TER 119- E14 wild-type (A) or JAK2-/- (C) fetal liver cells were transduced with a bicistronic retroviral vector (MIG) or MIG containing the indicated inserts and cultured in the presence or absence of Epo (2 U/mL) on RetroNectin. Thirty-six hours later, live GFP-positive cells from wild-type–(A) or JAK2-/--transduced cells (C) were assayed for CFU-E–generated colony content as determined by diaminobenzidine staining of hemoglobin. Graphs are from duplicates of 4 (A) and 3 (C) independent experiments. The average of the absolute number of CFU-E–derived colonies formed from vector control–transduced cells in the presence of Epo was 3160 ± 64 (n = 4) per 105 wild-type TER 119- cells and 1248 ± 263 per 105 JAK2-transduced JAK2-/-- (n = 3) plated cells. (B) Western blot analysis of transduced NIH 3T3 cells with 1:10 dilution of the retroviral supernatant. The upper band in the activated AKT*-transduced cells corresponds to the Flag-tagged myristylated AKT. (D) Real-time PCR analysis of the β-globin gene in live GFP-positive cells from panel A. Representative graph from duplicate analysis of 2 independent experiments. (E) Real-time PCR analysis of kinetics of up-regulation of β-globin gene expression in TER 119- E14 wild-type fetal liver–derived cells transduced with MIG-AKT* (results expressed as relative to time zero are from total cells). (F) JAK2-/- fetal liver–transduced cells with MIG, MIG-JAK2, or with a constitutively active AKT (MIG-AKT*) and cultured in the presence of Epo (MIG-JAK2 cells) or in the absence of Epo (vector control and MIG-AKT* cells) were analyzed for erythroid-cell differentiation 36 hours later (note TER 119+ cells within transduced GFP-positive cells). (G) Representative field of Wright-Giemsa staining of live GFP-positive transduced JAK2-/--derived cells (magnification, × 1000). Arrows show (i) polychromatophilic erythroblast and (ii) polychromatophilic erythroblast and normoblast. Images were obtained using a Nikon Eclipse E600 microscope (Nikon, Garden City, NJ) and a 100 ×/0.3 numeric aperture oil immersion lens. Images were captured using an RT Slider SPOT 2.3.1 camera and SPOT Advanced software (both from Diagnostic Instruments, Sterling Heights, MI).

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