Fig 2: 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. β-actin was used as loading control. dox, doxycycline. Source data are provided as a Source Data file

Fig 3: 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 4: Loss of ARID1A promotes adrenergic-to-mesenchymal transition and increases cisplatin resistance in NGP cells.(A) H3K27ac and BRG1 ChIP-seq tracks and RNA-seq tracks at PHOX2B and FN1 loci in control (red), ARID1A-/- #1 (blue), and ARID1A-/- #2 (black) NGP cells. (B) Western blot analysis of ARID1A, an adrenergic maker (PHOX2B), and three mesenchymal markers (FN1, SNAI2, and VIM) in control (Ctrl), ARID1A-/- #1, and ARID1A-/- #2 NGP cells. a-Tubulin was used as a loading control. (C) mRNA expression (RPKM of RNA-seq) of core regulatory circuitries of transcription factors specific for adrenergic (ADRN core TFs) or mesenchymal (MES core TFs) cells in control (Ctrl), ARID1A-/- #1, and ARID1A-/- #2 NGP cells. (D) GSEA to determine the enrichment of a generic gene signature for mesenchymal tumor in ARID1A mutant NGP cells. Genes are ranked by score and plotted along the x axis as vertical black bars. NES, normalized enrichment score; KO, knockout. (E) Cell viability [% relative to N,N'-dimethylformamide (DMF)–treated cells, y value] of control (red), ARID1A-/- #1 (blue), and ARID1A-/- #2 (black). NGP cells were measured after treatment with various concentrations of cisplatin (0 to 20,000 nM, x value) for 72 hours, and half-maximal inhibitory concentration (IC50) values are indicated. Values are means ± SD of triplicate experiments.

Fig 5: Analysis of B7-H3 mRNA expression in tumors and its clinical prognosis from TCGA database. (A) B7-H3 mRNA expression levels in resected ccRCC tissues and adjacent noncancerous tissue. (B) Kaplan–Meier analysis of disease-free survival as a function of high or low expression of B7-H3. (C) FN 1 mRNA expression levels in resected ccRCC tissues and adjacent noncancerous tissue. (D) Kaplan–Meier analysis of disease-free survival as a function of high or low expression of FN 1. *P < 0.05. TPM, Transcripts Per Kilobase of exon model per Million mapped reads.