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Fig. 2 Alcohol-induced nucleolar stress in mouse and chick CNCs is dose-dependent and persistent. (A, B) Dose-dependence of nucleolar stress. Alcohol reduced the nucleolar area per nucleus in a dose-dependent manner, at concentrations as low as 60mM for O9-1 cells (for UBF; A) and 29mM for primary CNCs (for nucleophosmin; B). Values are the mean ± SEM from 4 (O9-1) and 8 (primary CNCs) independent experiments. (C, D) Timecourse of nucleolar stress. The nucleolar reduction is rapid and occurs within 2hr (O9-1; 80mM) and 6hr (primary CNCs; 60mM) following the addition of alcohol at each cell lineage’s calculated EC50. Moreover, the nucleolar abundance remains low in both populations for at least 24hr following the 2hr alcohol exposure. Values are mean ± SEM from 12 (O9-1) and 8 (primary CNCs) independent experiments. For all analyses, nucleoli were visualized using antibodies directed against UBF (O9-1) or nucleophosmin (primary CNCs), and nuclei were visualized using DAPI. Data were analyzed using one-way analysis of variance with Holm-Sidak multiple comparisons.