a, ACSS2 localizes to the cytoplasm in undifferentiated CAD neurons. ACSS2 was imaged by immunofluorescence microscopy in CAD cells (4′,6-diamidino-2-phenylindole (DAPI) and α-tubulin (α-Tub) immunostaining show nuclei and cytoplasm, respectively). b, ACSS2 localizes to the nucleus in differentiated CAD neurons. c, Western blot analysis of cytoplasmic (CE) and nuclear (NE) extracts from undifferentiated CAD cells (undiff.) and differentiated CAD neurons (diff.) for ACSS2, ACL and histone H3. Nuclear ACSS2 expression is higher in differentiated cells (p.d.u., procedure defined unit; t-test P = 0.002, n = 3, mean ± s.d.). d, ACSS2 knockdown reduces differentiation-linked upregulation of neuronal gene expression program. Scatter plot contrasts the fold-change fragments per kilobase of transcript per million mapped (FPKM) of induced genes (Extended Data Fig. 2c) between wild-type (WT) and ACSS2 knockdown (KD) cells. Marginal distributions show histogram and kernel density estimation. Ordinary least squares linear regression is displayed with 95% confidence interval. e, Western blot of lysates from differentiated CAD neurons that were infected with lentiviral control (WT) or ACSS2 knockdown vector (shACSS2) (quantification shown in Extended Data Fig. 1g; n = 3). f, ACSS2 knockdown greatly reduces gene upregulation. Quintiles of upregulated genes (red dots in Extended Data Fig. 2c) with the greatest fold-change increase in wild-type cells (grey). Corresponding gene quintiles depict fold-change FPKM in ACSS2 knockdown cells (green) (for each quintile, columns represent the mean induction value of genes; mean ± s.e.m.). g, ACSS2i treatment of differentiated CAD neurons results in reduced expression of differentiation-induced genes. All genes are plotted in order of fold-change in wild-type CAD differentiation, and z-scores were computed for ACSS2i treatment and control, representing upregulation as blue and downregulation as red (RNA-seq in 24-h ACSS2i-treated and DMSO-treated control neurons, genes removed with z-score <0.5). Scale bar, 10 μm (a, b).