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First published online 22 March 2005
doi: 10.1242/jcs.02295

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NACA is a positive regulator of human erythroid-cell differentiation

¹ UMR599 INSERM, 27 Blvd Leï Roure, 13009 Marseille, France
² Institut Cochin, INSERM U567, CNRS UMR 8104, Université Paris V, Département d'Hématologie, Maternité Port-Royal, 123 Blvd de Port-Royal, 75014 Paris, France
³ Genetics Unit, Shriners Hospital, 1529 Cedar Avenue, Montreal, Quebec, H3G 1A6, Canada
⁴ Institut Paoli Calmettes, 232 Blvd de Sainte Marguerite, 13273 Marseille Cedex 9, France
⁵ Hopital fondation Saint-Joseph, Maternité Ste Monique, Blvd de Louvain, 13009 Marseille, France

View larger version (18K): [in a new window]   Fig. 1. Selective persistence in NACA protein expression during erythroid versus mono/macrophagic differentiation of TF-1 cells. Western-blot analyses were performed on THP1 and U937 cells cultured under standard conditions or on TF-1 cells cultured either for 24 hours in the presence of PMA and GM-CSF or for 3-5 days in the presence of Epo. Cells were harvested at the indicated times of culture and cell extracts taken. 20 µg total protein extracts were separated by 9% SDS-PAGE and analysed by western blot. Membranes were probed with a polyclonal immune serum against NACA and then probed again with an antibody against the p85 subunit of the phosphoinositide-3-kinase.

View larger version (55K): [in a new window]   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).

View larger version (60K): [in a new window]   Fig. 3. Effect of suppression of NACA by RNAi on hemoglobin expression in TF-1 cells. (A) Confocal pictures of cells grown in Epo either mock-treated or treated with a specific siRNA for NACA or an irrelevant siRNA, and then stained. (bottom right) Phase-contrast magnification of the cells treated with specific siRNA for NACA (top right). (B) SDS-PAGE western blots of TF-1 cells grown in Epo either mock-treated or treated with a siRNA specific for NACA or an irrelevant siRNA. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose then probed with an antibody specific for NACA (bottom) or a monoclonal antibody specific for the p85 sub-unit of the PI3-kinase (top). (C) A benzidine-staining assay was performed on cells cultured for either 3 days or 7 days under various conditions. The pictures show representative microscope fields of benzidine-stained cells when mock-treated and then cultured for either 3 days or 7 days in the presence of Epo alone (no siRNA) or transfected in the presence of Epo with either an irrelevant siRNA (control siRNA) or a specific siRNA for NACA (NACA siRNA). Histogram represents the corresponding proportions (±s.d.) of benzidine-stained cells when mock-treated were then cultured in the presence of either Epo alone (1) or transfected in the presence of Epo and either an irrelevant siRNA (2) or a specific siRNA for NACA (3) obtained from three independent experiments. 200 total cells were counted in each of the three independent experiments.

View larger version (32K): [in a new window]   Fig. 4. Selective persistence of NACA production during erythroid versus megakaryocytic or granulocytic differentiation of CD34+ cells. (A) Western-blot analyses were performed on CD34+ cells: (0) immediately after immunomagnetic purification; (2) after culture for 2 days; (6) after six days under expansion conditions. Alternatively, the western blot was performed after culture under conditions optimized for the cell growth and maturation of: erythroid (days 7+2, 7+5, 7+7, where day 7 corresponds to the end of the first Epo-free phase of the culture; Epo was added at day 7 and additional day numbers correspond to the duration of culture in the presence of Epo); megakaryocytic (days 6, 7, 10, 12, 14); granulocytic (days 6, 7, 14). Cells were harvested at the indicated times of culture. 20 µg total protein extracts were separated by 9% SDS-PAGE and analysed by western blot. Membranes were probed with a polyclonal immune serum against NACA and then probed again with an antibody against the ubiquitously expressed Grb2 adaptor. A result typical of those obtained from three different cell cultures is shown. Arrows indicate the molecular-weight markers. (B) Flow-cytometry analysis of CD34, GPA, CD42b, CD61 and CD15 surface-marker expression on cells either immediately following CD34+ purification (D0) or after 6 days of culture (D6) in expansion conditions, or after 10 days of culture in conditions optimized for erythroid, megakaryocytic or granulocytic growth and maturation, respectively. Bold profiles represent the fluorescence distribution of an isotype-matched control antibody. (C) Quantitative real-time RT-PCR analysis of NACA expression performed on mRNA from CD34+ cells either after 3 days or 6 days of culture under expansion conditions or after 10 days of culture under conditions optimized for erythroid, megakaryocytic and granulocytic growth and maturation, respectively. The expression of NACA transcript is expressed as a ratio relative to the expression of GAPDH.

View larger version (28K): [in a new window]   Fig. 5. Detection of ectopic expression of either NACA (S-tagged-NACA) or EGFP proteins or transcripts in transduced CD34+ cells cultured under erythroid and granulocytic differentiation conditions. 72 hours after the initiation of the transduction with NACA or EGFP lentiviral vector, CD34+ progenitor cells were cultured for 10 days under conditions optimized for erythroid or granulocytic cell growth and maturation. Cells were then harvested and the presence of the proteins or transcripts was examined. (A) Ectopic expression of NACA or EGFP proteins. The proteins from cell lysates, either enriched on protein-S beads (for S-tagged-NACA protein detection) or not (for the EGFP protein detection) were detected by western-blot analysis with either an anti-NACA or an anti-EGFP immune serum. Lysates were tested for their total protein content using an antibody against the Grb2 adaptor protein. (B) Time-course analysis of EGFP expression during granulocytic differentiation of CD34+ infected cells. Cells were cultured for up to 10 days under conditions optimized for granulocytic growth and maturation. The EGFP expression was analysed at 3 days and 10 days of culture by flow cytometry. Positive EGFP fluorescence (gate M1) is set according to the untransduced control cells. `%' indicates the proportion of EGFP-positive cells. (C) PCR Detection of genomic integrations of the lentiviral TRIPU3-EF1-S-tagged-NACA vector in the granulocytic cells. PCR was performed on cells collected after 10 days of culture under conditions that encourage granulocytic growth and maturation using a sense primer in the EF1 promoter and an antisense primer at the 3' end of the NACA-encoding cDNA. (D) RT-PCR detection of the transcript encoding S-tagged-NACA in granulocytic cells. RT-PCR was performed on cDNA templates obtained from cells collected after 10 days of culture in conditions that encourage granulocytic growth and maturation using a sense primer in the S-tagged sequence and an antisense primer in the 3' end of the NACA sequence. (C,D) Products were electrophoresed on 1.2% agarose gels and visualized by ethidium-bromide staining. `Mix' represents the negative control without template. Gr, granulocytic cells.

View larger version (25K): [in a new window]   Fig. 6. Flow-cytometry analysis of GPA surface expression in NACA- and EGFP-transduced CD34+ cells along differentiation of the erythroid lineage. 72 hours after the initiation of transduction, CD34+ cells were cultured under conditions optimized for erythroid-cell growth and maturation. Cell suspensions were stained at the indicated days of culture [day 5 (D5), day 7 (D7) and day 9 (D9)] with an anti-GPA-PE mAb and analysed by flow cytometry for cell-surface expression of GPA. (A) Unfilled profiles represent GPA expression by EGFP-transduced cells and filled profiles represent GPA expression by NACA-transduced cells. Isotype-matched control mAb was used in all experiments to set the parameters for analysis. Positive GPA fluorescence (gate M1) is set according to the isotype-matched control mAb. One representative experiment out of three performed is shown. (B) Results are expressed as the proportion of GPA cell-surface-marker-positive cells present in the cell culture.

View larger version (48K): [in a new window]   Fig. 7. Morphological characterization of either NACA- or EGFP-transduced CD34+ cells cultured in erythroid differentiation conditions. Pictures represent May-Grunwald-Giemsa staining of either EGFP- or NACA-transduced cells cultured for either 7 days or 12 days under erythroid differentiation conditions. The table indicates the proportion of proerythroblasts and basophilic, polychromatophilic and acidophilic erythroblasts in these transduced cells. Different cell types were quantified after counting a total of 200 cells per experiment (mean of three different experiments).

View larger version (24K): [in a new window]   Fig. 8. Effect of siRNA on the GPA cell-surface expression of CD34+ cells cultured in erythroid differentiation conditions. CD34+ progenitor cells were cultured for 3 days under conditions optimized for erythroid-cell growth and maturation in the presence of either an siRNA specific for NACA or an irrelevant siRNA. The GPA cell-surface marker was analysed by flow cytometry in the absence of siRNA duplexes (left), in the presence of an siRNA specific for NACA (center) and in the presence of an irrelevant siRNA (right). The numbers (insert) correspond to the proportion of GPA-positive cells.