Fig 2: Circ-CUX1 exerts an oncogenic role in tumorigenesis and aggressiveness A, BSoft agar (A) and Matrigel invasion (B) assays showing the anchorage-independent growth and invasion capability of IMR32 and SH-SY5Y cells stably transfected with empty vector (mock), circ-CUX1, scramble shRNA (sh-Scb), or sh-circ-CUX1 (n = 5). Scale bars: 100 µm. Student's t-test, one-way ANOVA, *P < 0.05 versus mock or sh-Scb.CRepresentative fluorescence images, in vivo growth curve, and weight at the end points of subcutaneous xenograft tumors formed by IMR32 cells stably transfected as indicated in nude mice (n = 5 for each group). Student's t-test, one-way ANOVA, *P < 0.05 versus mock or sh-Scb.D, EImmunohistochemical staining showing the expression of Ki-67 and CD31 (D) and glucose uptake, lactate production, and ATP levels (E) within subcutaneous xenograft tumors formed by IMR32 cells stably transfected as indicated (n = 5 for each group). Scale bars: 100 µm. Student's t-test, **P < 0.01 versus mock or sh-Scb.FRepresentative images, HE staining (arrowheads), quantification of lung metastatic colonization, and Kaplan–Meier curves of nude mice treated with tail vein injection of IMR32 cells stably transfected as indicated (n = 5 for each group). Scale bar: 100 µm. Student's t-test, **P < 0.01 versus mock or sh-Scb. Log-rank test for survival comparison.Data information: Data are presented as mean ± SEM. Exact P values are specified in Appendix Table S4.

Fig 3: Circ-CUX1 is up-regulated and enhances CUX1 expression in NB ASchematic illustration showing the generation of circ-CUX1 from CUX1.BRT–PCR or PCR assay revealing the amplification of circ-CUX1 from cDNA or genomic DNA (gDNA) of IMR32 and HeLa cells, with validation by Sanger sequencing.C, DReal-time qRT–PCR (C, normalized to ß-actin, n = 6) and Northern blot (D) indicating the circ-CUX1 levels in cell lines and IMR32 cells transfected with empty vector (mock) or circ-CUX1 and treated with RNase R (3 U µg-1). One-way ANOVA, *P < 0.05 versus HEK293.E, FReal-time qRT–PCR (E, normalized to ß-actin) and RNA-FISH with antisense junction probe and RNase R (3 U µg-1) treatment (F) showing the distribution and localization (arrowheads) of circ-CUX1 in IMR32 cells stably transfected with mock or circ-CUX1 (n = 5), using GAPDH and U1 as controls. Scale bar: 10 µm.GReal-time qRT–PCR assay indicating circ-CUX1 expression (normalized to ß-actin) and its correlation with CUX1 levels (Pearson's correlation coefficient) in tumor tissues, normal fetal adrenal medulla (FAM), or normal counterparts. Student's t-test, **P < 0.01 versus FAM or normal.HWestern blot showing the CUX1 levels in tumor cells stably transfected as indicated.Data information: Data are presented as mean ± SEM. Exact P values are specified in Appendix Table S4.

Fig 4: Circ-CUX1 directly interacts with EWSR1 protein in NB cells ASchematic illustration, Coomassie Blue staining, and Venn diagram showing the differential proteins pulled down by biotin-labeled linear or circular exogenous circ-CUX1, sense (S) or antisense (AS) probe targeting junction site of endogenous circ-CUX1 from IMR32 cells, and over-lapping analysis with RBP and proteins interacting with potential transcription factors of p200 CUX1 revealed by Genomatix program and BioGRID database.BWestern blot (upper panel) and RT–PCR (lower panel) assays indicating the proteins and transcripts pulled down by biotin-labeled linear or circular exogenous circ-CUX1, sense (S) or antisense (AS) probe targeting junction site of endogenous circ-CUX1 from IMR32 cell lysates, using AS probes of p200 CUX1 or CASP as controls.CRIP and real-time qRT–PCR assays revealing the interaction of EWSR1 with circ-CUX1, p200 CUX1, or CASP in SH-SY5Y cells and those stably transfected with empty vector (mock) or circ-CUX1 (n = 5). Student's t-test, *P < 0.05 versus IgG; ? P < 0.01 versus mock.D3D confocal images of dual RNA-FISH and immunofluorescence staining assay showing the co-localization of circ-CUX1 and EWSR1 in IMR32 cells stably transfected with mock or circ-CUX1. Scale bar: 10 µm.ERNA EMSA determining the interaction between biotin-labeled circ-CUX1 probe and EWSR1 protein within nuclear extracts of SH-SY5Y cells (arrowheads).F In vitro binding assay depicting the recovered circ-CUX1, p200 CUX1, or CASP detected by RT–PCR (upper panel) after incubation with GST-tagged recombinant EWSR1 protein validated by Western blot (lower panel).Data information: Data are presented as mean ± SEM. Exact P values are specified in Appendix Table S4.Source data are available online for this figure.

Fig 5: CUX1 facilitates aerobic glycolysis and tumor progression in NB AVenn diagram indicating the identification of differentially expressed glycolytic genes and transcription factors (fold change > 2.0, Student's t-test, P < 0.05) in 88 NB cases (GSE16476), and over-lapping analysis with potential transcription factors regulating glycolytic genes revealed by Genomatix program.B, CReal-time qRT–PCR (B, normalized to ß-actin, n = 5) and Western blot (C) assays revealing the expression of CUX1 and glycolytic genes in IMR32 cells stably transfected with empty vector (mock), p200 CUX1, scramble shRNA (sh-Scb), sh-CUX1 #1, or sh-CUX1 #2. Student's t-test, one-way ANOVA, *P < 0.05 versus mock or sh-Scb.D, EChIP and qPCR using Flag and CUX1 antibodies (D) and dual-luciferase (E) assays indicating the p110 CUX1 enrichment and promoter activity of ENO1, GPI, and PGK1 in IMR32 and SH-SY5Y cells stably transfected with mock, Flag-tagged p110 CUX1, sh-Scb, sh-CUX1 #1, or sh-CUX1 #2, and those treated with E64D (10 µM) for 24 h (n = 5). Student's t-test, one-way ANOVA, *P < 0.05 versus mock, sh-Scb, or DMSO.F, GSeahorse tracing curves (F), ECAR and OCR (G) of IMR32 cells stably transfected with mock, p110 CUX1, sh-Scb, sh-CUX1 #1, or sh-CUX1 #2, and those treated with E64D (10 µM) for 24 h (n = 5). Student's t-test, one-way ANOVA, *P < 0.05 versus mock, sh-Scb, or DMSO.Data information: Data are presented as mean ± SEM. Exact P values are specified in Appendix Table S4.Source data are available online for this figure.