Fig. 2. Enforced expression of NACA induces erythroid differentiation of TF-1 cells in the absence of Epo. TF-1 cells were transfected with NACA-IRES-EGFP vector plasmid and cultured for 48 hours in the presence of GM-CSF. Cells were then sorted by FACS (10⁵ EGFP-positive and EGFP-negative cells were collected) and benzidine stained. (A) Fluorescence imaging showing 2% of the EGFP-positive cells obtained after transfection with the NACA-IRES-EGFP vector plasmid. (B) Benzidine staining of sorted cells (EGFP-positive and EGFP-negative) performed 24 hours after EGFP sorting. Hemoglobinized cells stain dark blue. The proportion of benzidine was quantified after counting 200 cells. 80% of the sorted cells were benzidine stained (mean from three different experiments).