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PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The impact of varying proline and arginine concentration was assessed using RPMI free of arginine and proline and supplemented with 0%, 1%, 5%, 30%, 50% and 100% of standard RPMI levels of proline (174 μM) and arginine (1,149 μM).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For polyamine supplementation, putrescine, spermidine or 1-methyl-spermidine (BenchChem), were supplemented in the respective concentrations as described above along with 1 mM aminoguanidine (Sigma-Aldrich).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Additional rescue experiments were performed in different neuroblastoma cell backgrounds (SHEP, SKNBE2 and SHSY5Y).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For quantification cells were then stained with 10 μM Hoechst 33342 (Invitrogen) and 1 μg ml propidium iodide (Invitrogen) for 15 min at 37 °C/5% CO2.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Fluorescent signals were captured and analysed at different time points using the Perkin-Elmer Operetta system to generate growth curve.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Lentiviral particles were produced in HEK293 cells by co-transfection of lentiviral packaging plasmids pCMV-VSV-G and pPAX2 (Addgene) with pRSIT-U6Tet-shTarget-PGK-TetRep-2A-TagGFP2-2A-Puro (Cellecta) expressing the desired shRNAs using 25 kDa linear polyethylenimine (Polysciences).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Viral supernatants were collected 48 h post-transfection.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
IMR5 cells were transduced with viral supernatants containing 4 μg ml polybrene (Sigma) for 48 h. Cells were selected with 2 μg ml puromycin (Gibco) 48 h post-transduction.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
50 ng ml doxycycline (Sigma) was used for at least 24 h to induce shRNA expression.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The construct targets are reported in Supplementary Table 3.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cells were seeded into 12-well plates at the density of 2 × 10 cells per ml and induced with 50 ng ml doxycycline for 5 days.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cells were collected and incubated for 1 h in growth medium containing 10 μg ml Hoechst 33342(Invitrogen).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The stained cells were then analysed by flow cytometry and cell cycle distribution was quantified using the Modfit software.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
IMR5 cells were treated with 500 μM DFMO in 20% ProArg RPMI medium supplemented with 10% dialysed FBS for 5 days.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cells in log growth phase (< 80% confluence) were labelled with 1 μM puromycin at 37 °C for 1 h, then the medium was replaced by Versene (Gibco) and 5 μM cycloheximide (Sigma) to inhibit protein translation.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For western blot analysis lysates were separated electrophoretically and transferred to PVDF membranes (Bio-Rad), blocked with 5% non-fat milk in TBS-T, and detected with a mouse monoclonal puromycin antibody (Millipore, 1:10,000) at 4 °C overnight, followed by horseradish peroxidase-conjugated goat anti-mouse secondary (Proteintech, 1:10,000) for 1 h at room temperature.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Protein bands were visualized using enhanced chemiluminescence (ECL) reagent (Bio-Rad) and imaged on a ChemiDoc system (Bio-Rad).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For flow cytometry analysis, cells were fixed in 4% formalin at room temperature for 10 min and permeabilized with 0.1% Triton X-100 for 10 min.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cells were stained with PE anti-puromycin (BioLegend) for 30 min at 4 °C and analysed on a flow cytometer (Sony).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
After cell lysis samples were transferred to PVDF membranes as described above.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Antibodies used were used in the following concentrations: ODC1 (1:1,000, Abcam), MYCN (1:1,000, Santa Cruz), eIF5a (1:2,000, BD), eIF5A anti-hypusine (1:2,000, Merck Millipore), CENPR (1:1,000, Proteintech), KIF2C (1:1,000, Proteintech) and horseradish peroxidase-conjugated anti-GAPDH (1:10,000, Proteintech).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Total RNA from cells was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
One microgram of total RNA was used to generate cDNA adapted for quantitative PCR with reverse transcription (RT–qPCR) (TaKaRa Bio).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Real-time PCR was carried out using a Quant Studio 7 Pro Real-Time PCR machine (Applied Biosystems) and GoTaq qPCR Master Mix (Promega).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Fold change of gene expression was calculated by the 2 formula using GAPDH as an endogenous reference.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The list of primers for gene-expression analysis using RT–qPCR is reported in Supplementary Table 3.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Template creation: DNA constructs NheI_IRES_HA_EcoRI_xxx_BamHI and nLuc_Myc_EcoRV were synthesized by Gene Script and cloned with NheI and EcoRV sites into pcDNA3.1+ vector.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Triplet sequences were inserted between HA and nLuc using EcoRI and BamHI sites: 7× CCG-Pro and 7× CCA-Pro.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
DNA plasmids were linearized with NotI HF (NEB).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
In vitro T7 transcription with linear plasmids was performed with mMESSAGE mMACHINE Kit (Invitrogen, AM1344).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For the cell lysate preparation IMR5 cells were seeded in 15 cm dish at a density of 1 × 10 per dish and cultured for 5 days.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cells were then collected and centrifuged at 1,000g for 5 min at 4 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cell pellets (100 mg) were lysed in 1 volume (100 μl) of lysis buffer (LB, 30 mM HEPES/KOH (pH 7.6), 150 mM potassium acetate, 3.9 mM magnesium actetate, 4 mM DTT, 1% Triton X-100, 10% glycerol and protease inhibitor (Roche, 04693132001).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Four microlitres SUPERaseIn RNase inhibitor (Ambion, was added.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Cell debris was removed by centrifugation at 20,000g for 10 min at 4 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
RNA and protein concentration were quantified in the cell lysate.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Forty-microlitre aliquots of the cell lysates were snap frozen in liquid nitrogen and stored at −80 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Six microlitres were used for the in vitro translation reaction.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
In vitro translation was performed in a translational mix (15 mM HEPES/KOH (pH 7.6), 75 mM potassium acetate, 2 mM magnesium actetate, 1.75 mM ATP, 0.4 mM GTP, 50 μM complete amino acid mix (Promega, L4461), 20 mM creatine phosphate, 0.3 mg ml creatine kinase, 500 ng RNA) for 3 h at 35 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Spermidine was added in a final concentration 1.5 mM, if indicated.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Translation reaction product was detected with Nano-Glo Luciferase Assay System (Promega, N1110).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Collected Th-MYCN tumours were clamped and flash-frozen in liquid nitrogen.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
After this mechanical dissociation, crude protein extraction was obtained by lysis with CHAPS buffer (10 mM HEPES, 150 mM NaCl, 2% CHAPS) with fresh protease inhibitor and phosphatase inhibitor.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
This protein lysate (25 micrograms) was electrophoresed through a 5–10% Tris–glycine gel and immunoblotted using antibodies to MYCN (1:500, Cell Signaling), GAPDH (1:3,000, Cell Signaling Technologies) and eIF5A anti-hypusine (1:2,000, Millipore Sigma).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Crude protein extracts obtained as described above were electrophoresed through a slab isoelectric focusing gel (pH 3–7, Invitrogen Novex EC66452) with freshly made cathode and anode buffers (Novex).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The gel was transferred to a PVDF membrane and transferred using the iBlot transfer unit prior to blocking in buffer according to manufacturer’s recommendations for iBind.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The iBind was then assembled with a probe against eIF5A (1:3,000, BD Laboratories) and incubated for at least 2.5 h before developing.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Collected Th-MYCN tumours were preserved in 10% formalin and embedded in paraffin blocks.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Slides were cut and then stained with H&E. These slides were reviewed by a pathologist blinded to the treatment groups, and tumours were scored according to: (1) differentiation status; (2) neuropil presence or absence and relative abundance; and (3) evidence of global or localized necrosis.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Slides were then scanned and re-reviewed by the same pathologist.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Slides of formalin fixed, paraffin embedded tumours were stained on a Bond Max (MYCN, Ki67) automated staining system (Leica Microsystems).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The Bond Refine staining kit was used for MYCN and Ki67.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For MYCN (1:100, Abcam) and Ki67 (1:200, Abcam), the standard protocol was followed with the exception of the primary antibody incubation which was extended to 1 h at room temperature.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Antigen retrieval was performed with E2 (Leica Microsystems) retrieval solution for 20 min.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
After staining, all slides were rinsed, dehydrated through a series of ascending concentrations of ethanol and xylene, then cover-slipped.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Stained slides were then digitally scanned at 20× magnification on an Aperio CS-O or AT2 slide scanner (Leica Biosystems) and reviewed by a pathologist blinded to the treatment groups.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Tumours were collected and pulverized as described above.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
From the resulting tissue powder total RNA was extracted using TRIzol according to the manufacturer’s instructions (Invitrogen) followed by tRNA isolation by gel extraction from denaturing 8 M urea, 8% polyacrylamide gels.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Gel-extracted tRNA (560 ng) was enzymatically digested with 0.8 U nuclease P1 from Penicillium citrinum (Sigma, N8630) and 80 U GENIUS nuclease (Santa Cruz Biotech, sc-391121b) in 10 mM ammonium acetate (pH 6.0), 1.0 mM magnesium chloride at 40 °C for 70 min.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Hydrolysed ribonucleotides were dephosphorylated at 37 °C for 70 min by 0.4 U snake venom phosphodiesterase (Sigma, P3243) and 0.16 U alkaline phosphatase from Escherichia coli (Sigma, P5931) after adjusting the pH with ammonium bicarbonate to a final concentration of 50 mM. The hydrolysates were mixed with 3 volumes of acetonitrile and centrifuged (16,000g, 40 min, 4 °C).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The supernatants were lyophilized and dissolved in H2O for LC–MS/MS analysis.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Four biological replicates for each condition (CD, CD plus DFMO, ProArg-free, ProArg-free plus DFMO) were measured, each in two technical replicates.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Nucleosides were separated via reversed-phase chromatography using a Vanqush Neo UHPLC system (Thermo Fisher Scientific) and an Acquity nanoEase M/Z Peptide BEH C18 Column (130 Å, 1.7 μm, 300 μm × 150 mm; Waters, 186009259) and analysed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) operating in a positive-ion mode at a resolution of 45,000, the AGC target value set to 1.0 × 10 and the fill time to 50 ms.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Full MS spectra (m/z 244–576) and Top7 ddMS spectra with a nominal collision energy of 85% were recorded.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Quantitative analysis of LC–MS/MS data was performed using ElMaven software (https://github.com/ElucidataInc/ElMaven) and the identities of quantified ribonucleosides were verified by their specific fragmentation patterns in MS2 and by predetermined chromatographic elution orders of structural isomers.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Normalization was performed to the mean intensity of 32 nucleosides that were detected in all measurements.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For nucleosides that showed a marked difference in intensity between the two technical replicates due to degradation, the second technical replicate was excluded.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Tissues were disrupted by grinding in frozen state and lysed in lysis buffer (20 mM HEPES pH 7.2, 2% SDS).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Proteins extracts were diluted 1:1 with 2× SDS buffer (10% SDS, 100 mM Tris pH 8.5), boiled for 10 min at 95 °C, reduced with 5 mM (final) TCEP for 15 min at 55 °C, and alkylated with 20 mM (final) CAA for 30 min at room temperature.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Proteins were acidified by addition of 3% (final) perchloric acid, followed by addition of seven volumes of binding buffer (90% methanol, 100 mM TEAB).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Samples were loaded on S-trap columns and processed on a Resolvex A-200 positive pressure unit (Tecan).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Samples were washed 1× with binding buffer, 3× with 50% methanol/50% CHCl3 and 2× with binding buffer.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Digestion buffer (150 μl TEAB 50 mM) containing trypsin 1:10 (wt:wt, enzyme:protein) and Lys-C mix 1:50 (wt:wt, enzyme:protein) was added and incubated for 1 h at 37 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
One-hundred microlitres of digest buffer was added, and incubated overnight.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Peptides were eluted with 80 μl 0.2% aqueous formic acid followed by 80 μl of 50% acetonitrile containing 0.2% formic acid.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Peptides were diluted 1:1 with STOP buffer (PreOmics) and purified over iST positive pressure plates (PreOmics) according to the manufacturer’s instructions.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Peptides were separated on an Aurora (Gen3) 25 cm, 75 μm internal diameter column packed with C18 beads (1.7 μm) (IonOpticks) using a Vanquish Neo (Thermo Fisher Scientific) UHPLC.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Peptide separation was performed using a 90-min gradient of 2–17% solvent B (0.1% formic acid in acetonitrile) for 56 min, 17–25% solvent B for 21 min, 25–35% solvent B for 13 min, using a constant flow rate of 400 nl min.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Column temperature was controlled at 50 °C.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Mass spectrometry data were acquired with a timsTOF HT (Bruker Daltonics) in diaPASEF mode.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Mass spectrometry data were collected over a 100–1,700 m/z range.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
During each MS/MS data collection each PASEF cycle was 1.8 s. Ion mobility was calibrated using 3 Agilent ESI-L Tuning Mix ions: 622.0289, 922.0097 and 1221.9906.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
For diaPASEF we used the long-gradient method which included 16 diaPASEF scans with two 25 Da windows per ramp, mass range 400.0–1,201.0 Da and mobility range 1.43–0.6 1/K0.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The collision energy was decreased linearly from 59 eV at 1/K0 = 1.6 to 20 eV at 1/K0 = 0.6 V cm.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Both accumulation time and PASEF ramp time was set to 100 ms.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Raw mass spectrometry data were analysed with Spectronaut (v.17.1) in directDIA mode with standard settings.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Database search included the mouse Uniprot FASTA database.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Protein intensity values were normalized by log2 transformation and proteins with less than 70% of valid values in at least one group were filtered out.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The remaining missing values were imputed using the mixed imputation approach.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
In brief, missing values in samples belonging to the same group were imputed with k-nearest neighbours if there is at least 60% of valid values in that group, for that protein.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The remaining missing values are imputed with the MinProb method (random draws from a Gaussian distribution; width = 0.2 and downshift = 1.8).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
To measure the circulating turnover flux of a metabolite, we infused [U-C]labelled form of the respective metabolite via the jugular venous catheter.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
At pseudo-steady state, the fraction of the labelled of mass isotopomer form [M + i] of the nutrient in serum is measured as , such that i is from 0 to C and C is the total number of carbons in the metabolite.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The circulatory turnover flux Fcirc is defined as previously:1[12pt] $$_}}=R _}_}$$=R×1−L[M+C]L[M+C]where R is the infusion rate of the labelled tracer.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Since the turnover flux is a pseudo-steady state measurement, for minimally perturbative tracer infusions, production flux is approximately equal to consumption flux of the metabolite and thus Fcirc reflects both the circulating production and consumption fluxes of the infused metabolite in units of nmolC min g. The carbon atom circulatory turnover flux of the nutrient is calculated using2[12pt] $$_}}^}}=C R $$=C×R×1−LLwhere L is the fraction of labelled carbon atoms in the nutrient:3[12pt] $$L=_^i _}$$=∑i=0Ci×L[M+i]C measures the turnover of the whole carbon skeleton of the molecule, whereas measures the turnover of the carbon atoms in the molecule.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
When a [U-C]-labelled tracer X is infused, the normalized labelling of downstream metabolite Y is defined as , where LX and LY are the fraction of labelled carbon atoms for metabolite X and Y defined in equation (3).
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Direct fractional contribution of each metabolite to other metabolites in a tissue is calculated by setting up the follow set of linear equations:4[12pt] $$M (_}}\\ _}}\\ \\ _}})=(_}}\\ _}}\\ \\ _}})$$⋅fk←prolinefk←arginine⋮fk←glutamine=Lk←prolineLk←arginine⋮Lk←glutamineWhere is the fraction of k derived directly from i, M is the circulating metabolite interconversion matrix and is taken such that entry (X,Y) represents .
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Direct contributions to tissue were then calculated by performing an optimization procedure conditional on non-negative values by finding min ||M, f – L|| with respect to f such that f > 0.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
Standard error was estimated using a bootstrapping method (n = 100 simulations) by selecting values for M and L from normal distributions with means and standard deviations equal to calculated values for those parameters based on measured data.
PMC12527938
Reprogramming neuroblastoma by diet-enhanced polyamine depletion
The procedure for calculating fluxes between circulating nutrients has been previously described thoroughly.