MYCN protein, human

We tested medulloblastoma samples with the Illumina HumanMethylation450K DNA methylation array (Illumina, San Diego, CA, USA). The Gene Expression Omnibus accession number for 450K DNA methylation array profiles we used for the determination of human medulloblastoma molecular subgroup status is GSE93646.
To identify methylation-dependent subgroups, we did unsupervised class discovery by NMF-metagene and k-means clustering, testing all combinations of 3–10 metagenes and clusters for reproducibility using bootstrapped resampling methods (250 iterations) as described previously.⁷ (link) This analysis identified metagenes (a single score that reflects the methylation status of several CpG loci) representing the main biological effects present in the genome-wide dataset. We assessed cluster stability using the cophenetic index, a shorthand measure of the robustness of sample clustering as determined by consensus non-negative matrix factorisation (appendix p 3). We visualised clusters with t-SNE.²² We assigned samples classified with less than 80% confidence (by resampling procedures) as non-classifiable (NC; appendix pp 2–3).
We projected metagenes derived from our discovery cohort onto the validation cohort. Additionally, we combined the discovery and validation cohorts to do equivalent consensus clustering.
We assessed established medulloblastoma clinical, pathological, and molecular features as described previously.⁷ (link) Briefly, we defined histopathological variants according to the WHO 2016 guidelines.¹³ (link) We assigned metastatic status (M+) based on Chang's criteria (appendix p 3). Tumours were designated as R+ if their residuum after surgical excision exceeded 1·5 cm². Pathology was centrally reviewed by three experienced neuropathologists for 380 (89%) of 428 samples, and clinical data were collated from contributing centres and reviewed centrally (appendix p 3). We assessed MYC and MYCN status by fluorescence in situ hybridisation or copy-number estimates from methylation array. We assessed TP53, CTNNB1, and TERT mutation status by Sanger sequencing. We identified subgroup-specific differentially methylated CpG loci or methylated regions (DMRs) using limma or DMRcate23 (link), 24 (link) (appendix p 3). RNA-seq expression data were generated for discovery cohort samples for which mRNA of sufficient quantity and quality was available. We identified subgroup-specific differentially expressed genes using DESeq2,²⁵ (link) and these genes were included in ontology enrichment analyses (appendix p 4). We identified GFI1 mutations from RNA-seq data (appendix p 4).
MB_SHH mutation data were obtained from a previous study.²⁶ (link) Although 450K methylation data for MB_SHH subgroup assignment were not available for this sample cohort, the tightly defined age cutoff that we defined between the molecularly determined MB_SHH-Infant and MB_SHH-Child subgroups enabled us to infer subgroups for this sequencing cohort (appendix p 4).²⁶ (link) We tested recurrent MB_SHH mutations (TP53, SUFU, PTCH1, SMO, and TERT) and gene amplifications (MYCN and GLI2) identified by whole genome sequencing, for association with the age-defined MB_SHH-Child or MB_SHH-Infant subgroups using Fisher's exact test (appendix p 4).

SK-N-BE(2), IMR-32, and 293T cell lines were obtained from the American Type Culture Collection. Cell lines were authenticated by short tandem repeat profiling (GENEWIZ, Inc). SK-N-BE(2) and IMR-32 cells were maintained in a 1:1 mixture of Eagle's minimum essential medium and F12 medium (catalog nos.: 61100061/12500062; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (catalog no.: 10270106; Gibco), 100 units/ml penicillin, and 100 μg/ml streptomycin. 293T cells were cultured in Dulbecco's modified Eagle's medium (catalog no.: 10-013-CVR; Corning) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin.
The whole length of MYCN with a 3xFLAG tag sequence or 3xFLAG sequence only was constructed, subcloned into a Ubi-MCS-SV40-EGFP-IRES-puromycin vector, and further constructed as a lentivirus by Shanghai GeneChem Co, Ltd. 293T cells were treated with corresponding lentivirus for 24 h. After cultured for another 48 h, 2 μg/ml puromycin was used to screen stable infected cells for 24 h, and then the infection efficiency was determined by Western blot before further use. The sequences of full-length MYCN and four deletion mutated MYCN with a 3xFLAG tag (Δ1–123, Δ382–464, Δ346–464, and Δ281–464), and FLAG sequence only were constructed and subcloned into CMV-MCS-SV40-Neomycin vector. The amino acid sequence of 3xFLAG tag we used was DYKDDDDKGDYKDDDDKIDYKDDDDK. All plasmids were constructed and provided by Shanghai GeneChem Co, Ltd. These plasmids were transfected into 293T cells using Lipofectamine 2000 transfection reagent (Invitrogen) and transiently expressed according to the manufacturer's instruction. The transfection efficiency was validated by Western blot analysis of N-Myc expression.
Remodelin hydrobromide (S7641) was purchased from Selleck, and NU9056 (HY-110127) was purchased from MedChemExpress.

Control siRNA and three different siRNAs specific for EP300 and NAT10, respectively, were chemically synthesized (RiboBio, China). Cells were seeded in 6-well plates at 30 to 40% confluency 24 h before transfection. RNAi transfections were performed using RNAiMAX (catalog no.: 13778150; Thermo Fisher Scientific) following the manufacturer's protocol. Cells were harvested for subsequent experiments 48 to 72 h after transfection.
Target sequences were as follows:
EP300-1: 5′-CGACTTACCAGATGAATTA-3′
EP300-2: 5′-GCACAAATGTCTAGTTCTT-3′
EP300-3: 5′-AGATGAGAGTTTAGGCCGC-3′
NAT10-1: 5′-GGAATATGGTGGACTATCA-3′
NAT10-2: 5′-GGACTGCTGTAAGACTCTA-3′
NAT10-3: 5′-GTACTCCAATATCTTTGTT-3′
MYCN: 5′-CTGAGCGATTCAGATGATGAA-3′