Fig 2: High lncNB1, DEPDC1B, E2F1, and RPL35 in tumor tissues predict poor patient prognosis. a, b Two-sided unpaired Student’s t-test and two-sided Pearson’s correlation were employed to analyze the association between lncNB1 RNA expression and MYCN gene amplification (a) or DEPDC1B mRNA expression (b), respectively in 493 human neuroblastoma tissues in the publicly available RNA sequencing gene expression-patient prognosis SEQC-RPM-seqcnb1 dataset, downloaded from the R2 platform [http://r2.amc.nl]. In the box and whisker plot (a), the center line was the median, the ends of the box were the upper and lower quartiles, and the whiskers extended to the highest and the lowest 10% values. c, d Two-sided Pearson’s correlation was employed to analyze the correlation between RPL35 and DEPDC1B mRNA expression (c), as well as between E2F1 and DEPDC1B mRNA expression (d), in the SEQC-RPM-seqcnb1 dataset. e–h Kaplan–Meier curves showed the probability of overall survival of neuroblastoma patients according to the levels of lncNB1 (e), DEPDC1B (f), RPL35 (g), and E2F1 (h) expression in tumor tissues in the SEQC-RPM-seqcnb1 dataset, using the median of RNA expression as the cut-off points and two-sided log-rank test. i–l Kaplan–Meier curves showed the probability of overall survival of 181 stage 4 neuroblastoma patients according to the levels of lncNB1 (i), DEPDC1B (j), RPL35 (k), and E2F1 (l) in the SEQC-RPM-seqcnb1 dataset, using the median RNA expression as the cut-off points and two-sided log-rank test. Source data are provided as a Source Data file

Fig 3: LncNB1 is required for DEPDC1B mRNA and protein and E2F1 protein expression. a Genome-wide differential gene expression was examined with Affymetrix microarray in BE(2)-C cells 40 h after transfection with control siRNA, lncNB1 siRNA-1, or siRNA-2. Gene set enrichment analysis generated histograms confirming down-regulation of E2F1 target genes by lncNB1 siRNAs. FDR indicated false discovery rate. b, c BE(2)-C, Kelly, and CHP134 cells were transfected with control siRNA, lncNB1 siRNA-1, or lncNB1 siRNA-2 for 48 h. LncNB1, DEPDC1B, and E2F1 RNA expression was analyzed by RT-PCR (b), and DEPDC1B, E2F1, and N-Myc protein expression was analyzed by immunoblot (c). Data were shown as the mean ± standard error of three independent experiments, and evaluated by one-way ANOVA. *, **, and *** indicate P < 0.05, 0.01, and 0.001 respectively. d, e Doxycycline (DOX)-inducible control shRNA, lncNB1 shRNA-1 or shRNA-2 BE(2)-C (d) and Kelly (e) cells were treated with vehicle control or DOX for 48 h. RNA and protein were extracted from the cells for RT-PCR and immunoblot analyses of lncNB1, DEPDC1B, E2F1, and N-Myc. Data were shown as the mean ± standard error of three independent experiments, and evaluated by two-tailed unpaired Student’s t-test. *, **, and *** indicate P < 0.05, 0.01, and 0.001 respectively. Source data are provided as a Source Data file

Fig 4: LncNB1 stabilizes N-Myc protein through DEPDC1B-mediated ERK protein phosphorylation. a, b BE(2)-C, Kelly, and CHP134 cells were transfected with control siRNA, lncNB1 siRNA-1, lncNB1 siRNA-2 (a), DEPDC1B siRNA-1 or DEPDC1B siRNA-2 (b) for 48 h, followed by immunoblot analysis of DEPDC1B, ERK, phosphorylated (phos-) ERK, S62-phos-N-Myc, and total N-Myc protein expression. c BE(2)-C cells were transfected with control siRNA, lncNB1 siRNA-1, or DEPDC1B siRNA-1 for 30 h, and treated with 50 µM cycloheximide (CHX) for the last 15, 30, 45, or 60 min. Protein was extracted for immunoblot analysis of DEPDC1B and N-Myc. N-Myc protein level was normalized by actin, and N-Myc protein half-life (T1/2) was obtained from the line chart. d BE(2)-C cells were transfected with control siRNA, lncNB1 siRNA-1, or DEPDC1B siRNA-1 for 48 h, followed by treatment with MG-132 (10 µM) for 3 h. DEPDC1B and N-Myc protein expression was analyzed by immunoblot. e Doxycycline (DOX)-inducible control shRNA, lncNB1 shRNA-1 or lncNB1 shRNA-2 BE(2)-C and Kelly cells were treated with vehicle control or DOX for 48 h, followed by immunoblot analysis of DEPDC1B, phos-ERK, total ERK, S62-phos-N-Myc, or total N-Myc protein expression. Source data are provided as a Source Data file

Fig 5: LncNB1 is over-expressed in MYCN-amplified human neuroblastoma tissues and cells. a, b RNA was extracted from four MYCN-amplified (BE(2)-C, Kelly, CHP134, and SK-N-DZ) and two MYCN-non-amplified (SY5Y and SK-N-AS) neuroblastoma cell lines. Heatmap showed the top 40 genes most differentially expressed between the two groups of cell lines (a). Volcano plot revealed the top genes most significantly differentially expressed between the cell lines with the lowest false discovery rate (b). c lncNB1 RNA expression in human tumor tissues of various organ origins in The Cancer Genome Atlas (TCGA) cohort. The center line was the median, the bounds of the box were the upper and lower quartiles and the whiskers extended to 1.5× the inter-quartile range. Abbreviations of cancers were explained in the Methods section, and NB and SKCM represented neuroblastoma and skin melanoma. d RNA was extracted from MYCN-amplified and MYCN-non-amplified human neuroblastoma cell lines, followed by RT-PCR analysis of lncNB1 RNA expression. Data were shown as the mean ± standard error of three independent experiments. e, f lncNB1 and N-Myc RNA expression was extracted from the publicly available Maris-41-FPKM-rsg001 RNA sequencing dataset from 39 human neuroblastoma cell lines downloaded from R2 microarray analysis and visualization platform [http://r2.amc.nl]. Correlation between lncNB1 and N-Myc RNA expression was analyzed by two-sided Pearson’s correlation (e). The center line was the median, the bounds of the box were the upper and lower quartiles, and the whiskers were set to the minimum and maximum values (p = 0.001, two-sided unpaired Student’s t-test). Data were shown as the mean ± standard error (f). Source data are provided as a Source Data file