a, Percentage of cells with nuclear staining in ACSS2 immunofluorescence experiments (undiff., undifferentiated CAD cells; diff., differentiated CAD neurons; hippocampal, primary hippocampal neurons day 7; a minimum of 50 cells were examined in three replicate immunofluorescence experiments; t-test undiff. vs diff. P < 0.0001, undiff. vs hippocampal P < 0.0001; error bars, s.e.m.). b, Western blots of cytoplasmic (CE) and nuclear (NE) extracts from undifferentiated CAD cells and differentiated CAD neurons were probed with the indicated antibodies. c, d, Immunofluorescence in primary cortical neurons isolated from C57BL/6 embryos, at day 7 (c) and day 14 (d) of in vitro differentiation culture. ACSS2 locates predominantly to nuclei in differentiated primary cortical neurons. All scale bars, 25 μm. e, Immunofluorescence in primary hippocampal neurons isolated from C57BL/6 embryos at day 14 of in vitro differentiation culture. ACSS2 locates predominantly to nuclei in differentiated primary neurons. f, Immunofluorescence in primary hippocampal neurons at day 7 shows that ACL is chiefly localized to the cytoplasm. g, Neuronal differentiation markers decrease in ACSS2 knockdown cells. CAD cells were infected with lentiviral control (WT) or knockdown vector (shACSS2). Western blots of lysates from stably infected differentiated cells were probed with the indicated antibodies and quantified using ImageJ (n = 3; error bars, s.e.m.).