Fig. 5: NADs alter glioblastoma neurophysiology and engage an anti-proliferative AP-1/BTG GRN.
a, Workflow for DRUG-seq43 of drug-treated LN-229 cells. b, Transcriptional response of PCY-hit NAD-treated cells compared to NEG-treated cells (6 h; as in a). Significant genes by two-tailed Wald test (DESeq2) in light gray or colored according to their gene annotations (see legend). c, TFBS enrichment analysis of significantly upregulated genes in b. Circles, TF annotations. d, log2(fold change) of AP-1 TF and BTG family gene expression (columns) significantly upregulated by 6-h PCY-hit NAD (rows) treatment compared to NEG. e, Calcium response (ΔF/F0; y axis) over time (x axis) of LN-229 cells upon drug treatment. Timeline depicts FLIPR assay setup. Representative traces showing ΔF/F0, change in fluorescence intensity relative to baseline for NAD (left) and ONCD (right) drug conditions. f, Fold change in extracellular calcium influx upon drug treatment relative to DMSO measured as in e (n = 8 assay plates; n = 17 conditions; n = 18–30 wells per drug; DMSO, n = 47 wells). Asterisks in parentheses, median [Ca2+ fold change] < 0. Black line, median value. g, Single-cell-resolved calcium response (ΔF/F0) measured by ratiometric Fura-2 imaging over time at baseline (BASE) and after vortioxetine treatment (+VORT; 20 µM) across six cell lines (n = 3,561 cells; see also Extended Data Fig. 7c–f). Panels depict single-cell calcium responses (rows) over time (columns), stratified by the presence (Ψ) or absence (Ø) of calcium oscillations at baseline and VORT treatment. Representative single-cell traces (n = 4 per heatmap) are depicted below. h, Percent of cells displaying calcium oscillations (x axis) at baseline (gray) and after VORT treatment (purple) across cell lines (y axis; n = 6). Dots, independent experiments (n = 4–6 experiments per line). Paired two-tailed t-test. i, BTG1/2 transcriptional regulation (PathwayNet54). Black nodes, query genes; gray nodes, top 13 inferred TF interactions. Edge colors, relationship confidence. j, LN-229 confluency by live-cell imaging (y axis) over time (x axis) after gene knockdown. Mean (line) and standard deviation (bands) of n = 4 replicate wells are shown. k, LN-229 cell counts (y axis) after gene knockdown (columns) at baseline (left) and vortioxetine treatment (10 µM; right; n = 9–14 replicate wells per condition, n = 2 experiments). Normalized to FLUC at baseline. a,e,f, Drug abbreviations are in Supplementary Table 2. f,k, Two-tailed t-test. P values were adjusted for multiple comparisons by Holm correction. l, Summary diagram by which NADs target glioblastoma. CRE, cAMP response element; CKI, cyclin-dependent kinase inhibitor; FKH, forkhead binding motif. Box plots as in Fig. 1b. NS, not significant; PCY-HIT, PCY-hit; PCY-NEG, PCY-negative.
