Effect of p19^INK4D overexpression on cell cycle and CD41 or CD42 expression. (A) Validation of a lentiviral vector encoding a p19^INK4D cDNA (cDNA p19). Immunoblot analysis of p19^INK4D protein in 293T cell line transiently transfected with either the control vector or the p19 cDNA lentiviral vector. HDAC-1 was used as an internal control for protein quality and loading. (B) Immunoblot analysis shows that p19^INK4D overexpression induced a 2-fold decrease in phosphorylation of Rb on serine 780 (p-Rb). (C-D) CD34⁺ cells were transduced at day 1 and 2 of culture with the control or the p19 cDNA lentiviral vectors and GFP⁺ cells were analyzed at day 9. The MK population positive for CD41 and CD42 (C left panel) was analyzed for ploidy level by Hoechst staining (C right panel). The mean ploidy (N) was calculated from the number of cells of each ploidy level. (D) The mean fluorescence intensity (MFI) of CD41 (left panel) and CD42 (right panel) was analyzed separately for each ploidy class (from 2N to 32N). Data in panels C,D represent analysis 1 of 3 repeated experiments giving similar results. P values refer to 3 independent experiments.