Role of p15 and TGF-β1 in normal megakaryocytic differentiation.

(A) Neutralizing anti–TGF-β1 antibody induced a significant decrease of CD41⁺ cell number (*P < .001), without affecting total cell recovery. (B) p15 Amplification bands were detectable only in the presence of unmethylated sequence-specific primer pairs. Lanes 1 and 2, CD34⁺ cells; lanes 3 and 4, CD41⁺ cells on day 7; lanes 5 and 6, CD41⁺cells on day 14; lanes 7 and 8, negative control. U indicates unmethylated; M, methylated. (C) Semiquantitative RT-PCR analysis of p15 mRNA expression showed that p15 was not expressed in CD34⁺ cells (lane 1) and progressively increased in CD41⁺ cells on day 7 (lane 2) and day 14 (lane 4). The p15/β-actin ratios were 0.34 and 0.82 on days 7 and 14, respectively. Neutralizing anti–TGF-β1 antibody induced a significant reduction of p15 mRNA expression on day 7 (lane 3; p15/β-actin ratio, 0.14) and day 14 (lane 5; p15/β-actin ratio, 0.12). Negative and positive controls are shown in lanes 6 and 7, respectively. (D) Semiquantitative RT-PCR analysis of TGF-β1 mRNA expression demonstrated that TGF-β1 mRNA increases during megakaryocytic differentiation. TGF-β1/β-actin ratios were 0.25 in CD34⁺ cells (lane 1), 0.96 in CD41⁺ cells on day 7 (lane 2), and 2.63 in CD41⁺ cells on day 14 (lane 3). Negative and positive controls are shown in lanes 4 and 5, respectively.