Cell cycle and apoptosis analysis after Id1 knockdown and overexpression in fetal liver cells. (A) Representative cell cycle profiles after 24 and 48 hours of in vitro culture of lineage-negative E14.5 fetal liver cells transfected with knockdown constructs targeting Id1 or firefly luciferase. For clarity, the sub-G₁ population representing apoptotic cells has been gated out, but see supplemental Figure 4 for analysis of the sub-G₁ population. Bar charts summarizing cell cycle data (mean and SD) after 24 hours (B) and 48 hours (C) of culture from 1 of 2 independent experiments, each performed in triplicate and showing a similar result. Cdkn1a (D) and Cdkn1b (E) expression in lineage-negative E14.5 fetal liver cells cultured in vitro for 24 or 48 hours. In each case, data are normalized relative to expression in cells transfected with the knockdown construct targeting firefly luciferase at the 24-hour time point. (F) Representative FACS plots for 7AAD and annexin V staining in lineage-negative E14.5 fetal liver cells transfected with knockdown constructs targeting Id1 or luciferase after 48 hours of culture. Early apoptotic cells stain annexin V positive, 7AAD negative (gated population in the figure). (G) Bar chart summarizing the relative number of early apoptotic cells after transfection with knockdown constructs targeting Id1 or luciferase and 48 hours of in vitro culture. Summary data (mean and SEM) from 3 independent experiments, each performed in triplicate, are shown. Id1 knockdown is associated with a 2- to 4-fold increase in the proportion of early apoptotic cells (*P < .05; ***P < .001; see “Results” for details). (H) Bar chart summarizing the relative number of early apoptotic cells in lineage-negative E14.5 fetal liver cells grown in low erythropoietin levels and overexpressing Id1. Data are normalized relative to the number of early apoptotic cells in control (MIG) transfected fetal liver cells (*P < .05; see “Results” for details).