Fig 2: A transient phase of NOTCH3-IC expression induces commitment to a MES phenotype. a Western blot analysis of transient NOTCH3-IC induction. Time of doxycycline (dox) induction is indicated above the blots. Induction was followed by doxycycline wash-out and all samples were harvested after T = 14 days. Immunoblots show NOTCH pathway proteins (JAG1, NOTCH1, and NOTCH3), MES-markers (FN1 and YAP1) and ADRN-markers (PHOX2A, PHOX2B, DBH, and GATA2). As a positive reference, a sample of continued NOTCH3-IC induction for T = 14 days was run on the same gel (right). ß-actin was used as loading control. Source data are provided as a Source Data file. b Summed z-scores of mRNA signatures for MES and ADRN genesets. Non-induced control (-dox) cells are shown in dark blue. Persistent NOTCH3-IC induction of T = 7 and 14 days are shown in pink and red, respectively. A transient phase of NOTCH3-IC induction (T = 7 days + doxycycline, followed by T = 7 days - doxycycline wash-out) is shown in light blue. As a reference, various neuroblastoma cell lines are shown in gray dots and grouped by dashed lines according to MES and ADRN phenotypes (see ref. 1). c, d mRNA analysis (z-score of expression) of C) NOTCH pathway genes and D) core transcription factors of MES and ADRN lineages. + and—indicate culture conditions with or without doxycycline, respectively. Time of doxycycline treatment is indicated in days. For color-coding of samples, see panel c

Fig 3: NOTCH3-IC is a master regulator of ADRN-to-MES reprogramming a. Analysis of mRNA signature scores for MES and ADRN cells in a panel of neuroblastoma cell lines and a time series of NOTCH3-IC induction. Control (-dox) SH-SY5Y-NOTCH3-IC cells are depicted in blue; SH-SY5Y cells with induction of NOTCH3-IC (+dox) are shown in red. Time of NOTCH3-IC induction is indicated in days. Dashed circles group cell lines of MES- or ADRN-type as previously determined1. b Z-score mRNA expression of core super-enhancer associated transcription factors specific for ADRN or MES cells. Time of NOTCH3-IC induction (in days) is indicated above. dox, doxycycline. c Western blot analysis showing proteins of ADRN-markers (PHOX2A, PHOX2B, DBH, and TFAP2B), MES-markers (FN1, SNAI2, YAP1, and SOX9) and members of the NOTCH pathway (JAG1, NOTCH1, NOTCH2, NOTCH3, MAML2, and HES1) in SH-SY5Y with inducible overexpression of NOTCH3-IC. Time of NOTCH3-IC induction is indicated above the western blots. Protein lysates from 691-MES and 691-ADRN cells are loaded on the same blot as a reference for MES and ADRN states, respectively. ß-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file

Fig 4: NOTCH3-IC induces epigenetic remodeling of super-enhancer landscapes. a Analysis of H3K27ac marked super-enhancer regions (n = 1662) that were previously shown to be associated with MES or ADRN cells in neuroblastoma1. Shown are changes of H3K27ac signal in SH-SY5Y cells with induced expression of NOTCH3-IC (T = 7 days of induction). Red (increased) or blue (decreased) H3K27ac signal of the super-enhancers is depicted by horizontal bars on the genomic regions. Expanded views show the super-enhancer (SE) regions that distinguish MES and ADRN lines (indicated by orange and blue vertical bars, respectively) (right). b, c H3K27ac profiles in SH-EP2, SH-SY5Y and in SH-SY5Y cells with (NOTCH3-IC on) or without (NOTCH3-IC off) 7 days of doxycycline-induced expression of NOTCH3-IC. Shown are examples of b ADRN-specific SE-associated TF genes (ASCL1, PHOX2A) and ADRN lineage genes (DBH, DDC). In c, examples of MES-specific SE-associated TF genes (SMAD3, ELK4, NOTCH2, MAML2) are shown. SEs associated with ADRN- and MES cells are indicated by blue and orange horizontal bars, respectively. Gene color depicts orientation on sense (green) and anti-sense (red) DNA strands. d. Motif analysis on regions with increased H3K27ac signal. Statistical significance of enrichment was calculated using Fisher’s exact test with Bonferroni correction