Fig 2: Metabolic profiling of intracellular glycometabolism. Changes in metabolite levels after 48 h exposure to everolimus (closed bars, EVE) were compared with those without everolimus at 48 h (Control). The data represent the mean ± S.D. (n = 3). Statistically significant differences between the EVE and control: *p < 0.05; **p < 0.01 (Student’s t-test). Abbreviations: G6P glucose 6-phosphate, F6P fructose 6-phosphate, F1,6P fructose 1,6-bisphosphate, GAP glyceraldehyde phosphate, DHAP dihydroxyacetone phosphate, 1,3-Bis-PG 1,3-Bis phosphoglycerate, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, PEP phosphoenolpyruvate, Ac-CoA acetyl CoA, Ru5P ribulose 5-phosphate, R5P ribose 5-phosphate, Xu5P xylulose 5-phosphate, mTOR mammalian target of rapamycin, HIF1a hypoxia-inducible factor-1α, GLUT glucose transporter, HK1 hexokinase-1, GPI glucose-6-phosphate isomerase, PFKM 6-phosphofructokinase, ALDOA aldolase, TPI1P2 triosephosphate isomerase, GAPDH glyceraldehyde 3-phosphate dehydrogenase, PGK1 phosphoglycerate kinase-1, ENO1 enolase-1, PKM2 pyruvate kinase-2, LDHA_lactate dehydrogenase A, PDHA1 pyruvate dehydrogenase α1, G6PD 6-phosphate dehydrogenase, PGD 6-phosphogluconate dehydrogenase, RPIA ribose-5-phosphate isomerase A, TKT transketolase, TALDO1 transaldolase-1, CS citrate synthase, ACO1 aconitase-1, IDH1 isocitrate dehydrogenase-1, OGDH α-ketoglutarate dehydrogenase, DLST dihydrolipoamide succinyltransferase, SUCLG2 succinyl-CoA ligase, SDHA succinate dehydrogenase A, FH fumarate hydratase, MDH1 malate dehydrogenase-1

Fig 3: The interaction between PKM2 and P65.A The PKM2 promoter region had a binding box of P65. B Quantitative PCR analysis detected the chromatin–protein complex captured by CHIP P65 antibody containing the PKM2 promoter region sequence. *P < 0.05. C Western blotting analysis detected the whole-cell lysate protein captured by Co-IP P65 antibody containing PKM2 protein. D Western blotting analysis detected cell lysate protein captured by Co-IP PKM2 antibody containing P65 protein.

Fig 4: Effects of silencing pyruvate kinase muscle (PKM) splicers on expression profile of PKM isoform. A,C,E, Expression profile of PKM isoform after transfection with siRNA for PTBP1 (A), hnRNPA1 (C) or SRSF3 (E) for 72 h. Expression levels of PTBP1, hnRNPA1, SRSF3, PKM1, PKM2 and PKM1/2 proteins were examined by western blotting analysis. B,D,F, Ratios of PKM1/PKM2 after silencing PTBP1 (B), hnRNPA1 (D), and SRSF3 (F) were quantified by densitometry scanning. hnRNP, heterogeneous nuclear ribonucleoprotein

Fig 5: Analysis of vesicular proteins in the plasma of neuroblastoma patients. (a) The indicated proteins were identified in the cargo of EVs isolated from the plasma of patients with (MYCN A NB) or without (MYCN N/A NB) MYCN-amplified neuroblastomas by western blot analysis. Plasmas from non-cancer patients were used as controls. Gal-3BP was used as loading control and total cell lysates from TET21-N cells were used as a positive control for PKM and hexokinase II. * indicates non-specific band; ** indicates specific band. Kaplan–Meier survival curve correlating the expression of PKM (b) and hexokinase II (c) with event-free neuroblastoma patients' survival in the Kocak (i) and SEQC (ii) datasets available in the R2 genomic visualization platform (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi).