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.