Endogenous EKLF activates erythrocytic and represses megakaryocytic gene expression in MEL cells. 4D7 and 2M12 are 2 different clones derived from MEL cells that have been engineered to allow inducible expression of 2 different shRNAs directed against Eklf mRNA (“Cell lines culture and transfection”). 4D7 and 2M12 cells were grown for 2 days in the presence of 2 μg/mL doxycycline to induce Eklf shRNA expression and then for 2 days in the presence of 5 mM HMBA (still in the presence of doxycycline) to induce their differentiation. 4D7 and 2M12 cells grown in the same conditions but without doxycycline were used as control. (A) Western blot analysis of EKLF protein in 4D7 (left panels) or 2M12 cells (right panels) treated in the presence (lane 2) or absence (lane 1) of doxycycline. Asterisk indicates unspecific band. Actin protein is shown as loading control. Percentages indicate relative levels of EKLF proteins (EKLF/actin ratios) estimated by densitometry. (B) Doxycycline-induced changes of erythrocytic (lanes 1-3) and megakaryocytic (lanes 4-7) mRNA gene levels in 4D7 cells and 2M12 cells. Relative levels of each mRNA have been determined by quantitative reverse-transcription (RT)–PCR using Hprt mRNA as a normalization reference. Final results are expressed as fold changes induced by doxycycline (means and SD from 4 independent experiments).