Endogenous AP2A2 localization in hematopoietic and leukemic cells. All subsequent cells stained for AP2A2 (Cy3-red), NUMB (FITC-green), and nucleus (DAPI-blue). (A) Adult CD150⁺48⁻LSK cell showing disparate AP2A2 and Numb vesicles. (B) Localization of endogenous AP2A2 and NUMB in CD 150⁺48⁻LSK HSCs. Definitions were as follows: vesicles, if vesicles are clear and distinct (see panel A); clusters, if any vesicles are coalesced (see panel C); colocalized, if vesicles or clusters are yellow when merged (see panels A and C). Total number of cells, N = 75. Colocalization percentage for vesicles, n = 39 cells; colocalization percentage for clusters, n = 36 cells. Values represent means ± SEM from 2 independent experiments. (C) Adult CD150⁺48⁻LSK cells showing asymmetric AP2A2 and NUMB clusters that are (lower cell) or not (top cell) colocalized. (D) Comparison of AP2A2 distribution in adult HSCs (n = 120 from 3 independent experiments) versus E14.5 FL HSCs (n = 127 from 2 independent experiments). Asymmetric defined arbitrarily as distinctly more AP2A2 vesicles on half of the HSCs in any plane (panels A and E are symmetric; panels C and F asymmetric). Values represent means ± SEM. (E) E14.5 FL CD150⁺48⁻Sca⁺Mac1⁺Lin⁻ cells with symmetric distribution. (F) E14.5 CD150⁺48⁻Sca⁺Mac1⁺Lin⁻ cell exhibiting asymmetric AP2A2 with symmetric NUMB. (G) A representative FL E14.5 CD150⁺48–Sca⁺Mac1⁺Lin⁻ cell during mitosis showing endogenous AP2A2 and NUMB as diffusely symmetric vesicles that are essentially not colocalized (n = 7 cells in mitoses). (H) Still frames from live-cell videomicroscopy of representative adult CD150⁺48⁻LSK cell cotransduced with cherry-Ap2a2 (Cherry-red) and venus-Numb (FITC-green) constructs showing the absence of colocalization during mitosis. Total of n = 20 cells seen in mitoses. From 2 independent experiments, 17 of 20 cells (85%) seen in mitoses showed absence of colocalization. (I) FLA2 leukemia cells stained for Ap2A2 and NUMB. (J) Adult CD150⁺48⁻ LSK cells transduced with vector alone and (K) HoxB4, then stained for AP2A2, (see supplemental Figure 2B). All confocal images were acquired with an inverted LSM 510 microscope (Carl Zeiss) using Planapochromat 63×/1.4 numerical oil lens objective (Carl Zeiss) and analyzed with LSM Version 3.2 software (Carl Zeiss). All scale bars indicate 10 μm.