p19^INK4D expression in erythroid and megakaryocyte human cells. (A) The absolute expression level of p19^INK4D per 100 ng total RNA was measured relatively to different dilutions of plasmids (corresponding to 10² to 10⁶ copies) harboring a p19^INK4D cDNA. The graph represents the expression level of p19^INK4D in immature (CD36⁺GPA⁻) and mature (CD36⁺GPA⁺) erythroid cells and in mature (CD41⁺CD42⁺) MKs. The error bars represent the standard deviation of the mean of 3 experiments each performed in triplicate wells. (B-E) CD34⁺ cells were cultured with TPO for 9 days. Then, CD41- and CD42-positive cells stained with Hoechst were sorted on their ploidy level (from 2N to 16N). (B) mRNA levels of p15^INK4B, p16^INK4A, p18^INK4C, and p19^INK4D and their variants were quantified by real-time RT-PCR. Their relative expression was calculated in comparison with HPRT mRNA. (C) mRNA levels of p18^INK4Cv2 and p19^INK4D were quantified by RT-PCR in each ploidy class (from 2N to 16N) and normalized to their expression in diploid (2N) cell fraction. (D) The protein level of p19^INK4D in different ploidy fractions was measured relatively to HDAC-1, a protein that we found stable during ploidization. The graph represents the ratio between p19^INK4D and HDAC-1 levels for each ploidy class. The data illustrate 1 of 2 representative experiments that produced similar results. (E) To investigate the expression level of p19^INK4D during differentiation, total cell population was harvested on day 3 (D3), day 6 (D6), and day 9 (D9) and submitted to Western blot analysis. (F) The expression level of p19^INK4D mRNA was investigated by QRT-PCR in diploid (2N) cells sorted at day 6 in culture in 3 MK populations corresponding to very immature (CD34⁺CD41⁺CD42⁻), immature (CD34⁺CD41⁺CD42⁺), to more mature (CD34⁻CD41⁺CD42⁺) cells. The error bars represent the standard deviation of the mean of 3 repeated experiments each performed in triplicate wells.