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Fig. 1 Acute alcohol exposure causes nucleolar stress in the mouse CNC line O9-1 and in primary chick CNCs. (A-D) Murine O9-1 cells were exposed to 60mM ethanol for 2hr and assessed 6hr after the alcohol addition. Compared to untreated cells (Control, left panels), alcohol (middle panel) altered the distribution of nucleoli per nucleus such that there were fewer nucleoli in alcohol-exposed CNCs, as assessed by immunostain for the nucleolar proteins (A) fibrillarin, (B) nucleophosmin, (C) UBF, and (D) nucleolin. Values that quantify the distribution of nucleolar number per nucleus (right panels) are the mean ± SD of three independent experiments as detailed in Methods. (E-G) Primary chick CNCs, derived from cranial explants, were exposed to 60mM ethanol for 2hr and assessed 6hr after the alcohol addition. Compared to untreated cells (left panels), alcohol (middle panels) altered the distribution of nucleoli per nucleus such that there were fewer nucleoli in alcohol-exposed CNCs, as visualized using immunostain for (E) fibrillarin, (F) nucleophosmin, and (G) coilin. Values that quantify the distribution of nucleolar number per nucleus (right panels) are the mean ± SD for N = 3?7 experimental replicates as detailed in Methods. For all, nuclei were imaged using DAPI counterstain and representative images are shown; scale bar represents 50 ?m. Data were analyzed using chi-square analysis.