Cyclin D3 is functionally downstream of Meis1. (A top) Western blot analysis of U937+Meis1 and U937+M33-Meis1 cells stimulated with SCF and immunoblotted for hypophosphorylated (pRb) and hyperphosphorylated (ppRb) forms of Rb. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as the loading control. (Bottom) densitometry analysis quantifying ppRb. (B) Intracellular phospho-pRb staining of Hoxa9 and ND13 BM cells transduced with Meis1 or M33-Meis1. The cells were immunostained (shaded) or untreated (unshaded) with anti–phospho-pRb and analyzed by flow cytometry. The Kolmogorov-Smirnov chi-square value and the super enhanced D-max (SED %Positive) statistics are shown. (C top) Schematic representation of cyclin D3 showing the putative start site (arrow), exons (black boxes), 5′ and 3′ untranslated regions (unshaded boxes), and introns. The PCR amplicons are labeled C1, C2, and C3. (Bottom) Data represent the mean of fold enrichment relative to immunoglobulin G ± SD of 3 independent experiments. (D) ND13pac BM cells were cotransduced with M33-Meis1-YFP and cyclin D3-GFP, stained with Hoechst-pyronin, and analyzed by flow cytometry. (E) In vitro growth kinetics of indicated cells plated in standard growth conditions. The data are expressed as the mean ± SD of 2 independent transductions.