Fig 1: Multiple BET inhibitors enhance synaptic gene expression.(a) Schematic of mRNA-seq experiment using 3–6 replicates per treatment condition. hN + hpA co-cultures were treated with 0.1% DMSO, 0.5 µM (+)-JQ1 or 1 µM Birabresib for 72 hr prior to harvesting. (b, c) Volcano plots for (+)-JQ1 versus DMSO control (b) and Birabresib versus DMSO control (c). Log2 fold change is shown on the x-axis, with the -log10 of the adjusted p-value shown on the y-axis. Positive fold change reflects an increase in the treatment condition relative to DMSO control. Numbers of significantly differentially expressed genes (DEGs) are shown for each condition. (d) Overlap (shown as number of genes) between DEGs identified with (+)-JQ1 treatment (blue) and DEGs identified with Birabresib treatment (green). (e) Scatterplot of the fold change read counts of the genes upregulated by (+)-JQ1 and Birabresib. Note the strong positive Pearson’s r (94%) correlation between transcripts upregulated by (+)-JQ1 and Birabresib. Select gene examples are highlighted in red. (f) Select canonical pathways identified by IPA for DEGs upregulated in both (+)-JQ1 and Birabresib treatment conditions (top) or downregulated in both (+)-JQ1 and Birabresib treatment conditions (bottom). The x-axis shows -log10 of the p-value for each pathway analysis term. (g) SynGO analysis showing the top 5 significantly enriched cellular component and biological process ontology terms identified using all genes upregulated by (+)-JQ1 and Birabresib. The x-axis shows -log10 of the p-value for each ontology term; the top two terms also met a<1% FDR threshold (h) SynGO analysis sunburst representation showing gene counts of the cellular components identified using all genes upregulated by (+)-JQ1 and Birabresib. (i) Graphs of transcript expression values for pre- and post-synaptic genes. Significance was calculated by Benjamini–Hochberg adjusted Wald test as part of the DEseq2 RNA-seq experiment. n=3 biological replicates. (j) Western blot analysis of BAIAP2 and Homer1 protein expression levels following treatment with DMSO, (+)-JQ1, or Birabresib (n = 4 biological replicates). Top, quantification (shown as % of DMSO control); bottom, example blots. For all figure panels, significance is indicated by *p<0.05, **p<0.01, ***p<0.001 relative to controls. Figure 6—source data 1.Source of western blots for BAIAP2 and Homer1 as well as Cofilin loading control following DMSO, (+)-JQ1, or Birabresib treatment conditions.Arrowheads indicate bands used for quantification.