TET3 regulates terminal cell differentiation at the metabolic level

Generation of Tet3 knockout mice

A targeting vector introducing loxP sites around Tet3 exon 5 was constructed and electroporated into (C57BL6/J x C3H/HeJ) F1 embryonic stem cells (ESCs). Deletion of the floxed region results in a frame-shift that introduces a premature stop codon in exon 6, triggering nonsense-mediated decay of the mutant transcript. The construct also contained an FRT-flanked selection cassette coding for neomycin and thymidine kinase. Individual neomycin-resistant ESC clones were screened for recombination in the proper genomic locus. Next, FIAU-negative selection was used to select for clones that had excised the thymidine kinase cassette after transient transfection of an Flp-coding plasmid. Then, a Cre-coding plasmid was transiently transfected to excise the loxP-flanked exon 5 and single ESC clones were genotyped to confirm the excision. Tet3 knockout ESCs were used to generate chimeric mice through aggregation with morula stage (C57BL6/J x C3H/HeJ) F1 embryos. The generated male chimeric mice were crossed to CD1 females to screen for germ line transmission of the Tet3 knockout allele. Finally, Tet3 heterozygous females were backcrossed with pure C57BL6/JRccHsd males at least 5 generations to obtain an incipient congenic line (N5 or >95% C57BL6/JRccHsd). Genotyping primers are shown in Supp. Table 3. Mice were time-mated and the presence of a vaginal plug was considered as embryonic day (E) 0.5. Experiments were performed on E18.5 embryos obtained by caesarian section, as the gestation length of this particular mouse line is 19 days. Both male and female Tet3 knockout mice exhibit a perinatal lethal phenotype, thus sex was not considered in the study design for not being relevant in this phenotype. Animals were housed in a controlled environment with a 12 h light/dark cycle in open cages at 20-24 °C and 55–65% humidity, with access ad libitum to a standard chow diet and water. All animal experiments were performed in accordance with the animal care guidelines of the European Union (2010/63/EU) prior approval of the Spanish National Research Council Ethics Committee and the Valencian Regional Government (Approval reference: 2016/VSC/PEA/00187).

RNAscope in situ hybridization and quantification analysis

The first 2 cm of E18.5 mouse small intestines that include the whole duodenum and partial jejunum from wild-type and Tet3 knockout embryos were dissected, fixed in formalin and shipped to Charles River Laboratories Evreux. Then, the samples were embedded in paraffin, sectioned (3–5 µm thick), and stained with RNAscope probes (ACD). All images were stained with TET1 (probe 455228-C1), TET2 (probe 511598-C4), TET3 (probe 505498-C3) and one of the following cell-type specific probes: OLFM4 (311838-C2), MUC2 (315458-C2), CHGA (447858-C2) or VIL1 (463308-C2). The probe 321818 and the probe 321838 were used as positive and negative controls, respectively. The staining was performed on a Leica BOND instrument. All samples were scanned at 40x on an Akoya PhenoImager (Akoya Biosciences) and the generated qptiff files were transferred to PharmaServices team at Indica Labs for analysis. The HALO® v3.6.4 (Indica Labs) quantitative image analysis software and the HALO® FISH v3.2.3 algorithm was employed to detect and count the fluorescent RNAscope probes after cell segmentation.

Immunofluorescence, confocal microscopy and image analysis

E18.5 mouse small intestines were dissected, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 µm). Double stainings were performed first for 5hmC detection using the Tyramide Superboost kit (ThermoFisher Scientific) followed by stripping and second staining for 5mC. Antigen retrieval and antibody stripping was performed by incubating sections 25 min in sodium citrate buffer 10 mM pH 6 in a water bath at 95 °C. Antibodies are listed in Supp. Table 4. Images were acquired with a SP8 confocal microscope (Leica Microsystems) or a LSM980 with Airyscan2 (Zeiss) and processed using ImageJ v.1.6.0. Hematoxylin and eosin (H&E) staining was performed as previously reported⁴⁵. Whole embryo and H&E intestine section images were obtained with an Aperio VERSA Scanner (Leica Microsystems).

5hmC quantification

DNA was extracted from epithelial cells sorted from the whole E18.5 small intestine using the Qiamp DNA Mini kit following the manufacturer’s protocol. DNA was hydrolysed for 6 h at 37 °C using DNA degradase plus (Zymo Research). Mass spectrometry quantification was performed as previously described⁴⁶ in the Babraham Institute mass spectrometry facility using three individual E18.5 wild-type and three individual E18.5 Tet3 knockout embryos.

RT – quantitative PCR

RNA was extracted from villi isolated from the whole E18.5 small intestine using the Qiamp RNeasy micro kit, including the DNAse treatment step, following the manufacturer’s protocol. Quantitative PCR was performed in technical triplicates on a Quant Studio 5 Real-Time PCR System (Applied Biosystems) using Power up Sybr Green (Thermofisher) and a primer concentration of 375 nM. Primers are listed in Supp. Table 3. Actb and Gapdh were used as endogenous controls for gene expression analysis. Fold enrichment normalized to wild-type samples was calculated using the 2^−ΔΔCt method⁴⁷.

Intestine dissociation and cell sorting

A single cell suspension from whole mouse small intestinal epithelium was obtained following a previous reported protocol⁴⁸. Briefly, the small intestine was dissected from E18.5 embryos, opened longitudinally, and placed on ice-cold PBS containing 30 mM EDTA and 1.5 mM DTT for 20 min on ice. Next, the intestine was transferred to a tube containing 30 mM EDTA in PBS, incubated at 37 °C for 10 min and, then, vigorously shaken for 30 s to release the intestinal epithelium from the lamina propria. After removing the intact intestinal muscle, the villi suspension was used for mass spectrometry quantification and whole genome bisulfite sequencing or dissociated into single cell for fluorescence activated cell sorting. To generate a single cell suspension, the villi were incubated at 37 °C for 14 min in 10 ml HBSS (with Ca²⁺ and Mg²⁺) containing 8 mg of dispase II. During the incubation, the tube was vigorously shaken for 30 s every 2 min to dissociate the villi. Finally, 10% fetal bovine serum and 0.5 mg DNase I were added to the cellular suspension that was sequentially passed through 100 µm and 40 µm filters for obtaining single cells. The single cell suspension was incubated with CD31-PE, CD45-PE, Ter119-PE, EpCAM-APC780 fluorescent-labelled antibodies for 30 min. DAPI was used for staining dead cells. A BD FACS Aria Fusion (BD Biosciences) was used for sorting the live intestinal epithelial population (DAPI—negative, CD31-PE—negative, CD45-PE—negative, Ter119-PE—negative and EpCAM-APC780—positive cells). For all fluorescent channels, positive and negative cells were gated on the basis of fluorescent minus one control. Antibodies are listed in Supp. Table 4. For scRNA-seq, all steps were performed under RNase-free conditions and 0.5U/µl of RiboLock RNase inhibitor (ThermoFisher Scientific) was added to all buffers. Cell sorting was performed in the University of Valencia Flow Citometry Unit.

Single cell RNA-seq

Epithelial cells sorted from the whole E18.5 small intestine were fixed in 90% ice-cold methanol and stored at −80 °C until analysis. Tet3 knockout and wild-type samples correspond to a pool of intestinal epithelial cells from two embryos each. 6000 cells per sample were analyzed. For analysis, cells were resuspended in 3x saline sodium citrate buffer containing 0.04% BSA, 1% SUPERase·In RNAs Inhibitor (Invitrogen) and 40 mM DTT⁴⁹. Single-indexed libraries were prepared using the Chromium Single Cell 3’ v3/v3.1 chemistry (10x Genomics) following the manufacturer’s instructions. The generated libraries were sequenced on an Illumina Novaseq (Illumina) system using the following transcript read lengths: 28 bp for cell barcode and UMI, 8 bp for sample index and 91 bp for insert. 100,000 reads per cell were sequenced. scRNA-seq analysis was performed at Princess Margaret Genomics Centre (Toronto, Canada).

Single cell RNA-seq data analysis

For scRNA-seq data processing, the alevin pipeline integrated with the salmon software (version salmon-0.14.1) was used, performing cell barcode (CB) detection, read mapping, unique molecular identifier (UMI) deduplication, gene count estimation and cell barcode whitelisting⁵⁰. Reads were aligned and annotated to the mouse GRCm38 reference genome, transcriptome and annotation, downloaded from GENOME website (version 21). Alevin’s forceCells option was set to 6000 for both wild-type and Tet3 knockout populations to specify the number of CBs to consider for whitelisting. Standard procedures for filtering, variable gene selection, dimension reduction and clustering were performed using the Scanpy toolkit (version 1.7.1)⁵¹. Cells with fewer than 200 detected genes were removed from the analysis as well as cells with a mitochondrial versus all gene fraction greater than 0.15. Genes detected in more than 3 cells were considered as expressed, while genes with more than 100,000 total counts were excluded. The data matrix was total-count normalized to 10,000 reads per cell and stabilized by computing the natural logarithm of one plus the counts for each cell. Highly variable genes were filtered for feature selection, remaining a data matrix of 11871 cells (5933 WT and 5983 Tet3 knockout) and 1350 genes. Each gene was scaled to unit variance and values exceeding standard deviation 10 were clipped. For dimensional reduction and clustering, the t-Distributed Stochastic Neighbor Embedding (t-SNE)⁵² and the Louvain graph-clustering method²¹ were used. The ranking for the characterizing genes of each cluster was computed using the Wilcoxon test with Benjamini-Hochberg correction method. For trajectory inference, a partition-based graph abstraction (PAGA) map preserving the global topology of data was performed⁵¹ and single-cell graphs using ‘fa’ (ForceAtlas2)⁵³ layout and PAGA initialization were made, one for the Louvain cluster groups, and another for the wild-type and Tet3 knockout groups. Gene ontology analysis was performed using the g:Profiler web server with default parameters⁵⁴ using the top 100 gene markers for wild-type or Tet3 knockout specific clusters. Gene Set Enrichment Analysis (GSEA) was performed with GSEA v.4.1.0 desktop software using the GSEA Preranked tool with SIGN(log2FC)*(-log10(p-value)) as ranking parameter for all detected genes, where SIGN is the sign operator and FC the Fold Change, and c2.cp.kegg.v7.4.symbols.gmt as enrichment gene set⁵⁵.

Transmission Electron Microscopy

Small intestines were obtained from Tet3 knockout and wild-type 18.5 d.p.c. embryos by caesarian section. The first two centimeters of the small intestine that include the entire duodenum and jejunum were immediately fixed overnight at 4 °C by immersion in 0.1 M phosphate buffer pH 7.4, containing 4% paraformaldehyde and 2% glutaraldehyde, followed by postfixation with 1% osmium tetroxide- 0,8% potassium ferrocyanide mixture in water for 1 hour at 4 °C. Samples were then dehydrated with ethanol and propylene oxide and embedded in Epon resin. Semi-thin sections were obtained from the resin blocks and stained with toluidine blue to identify regions of interest in intestine and heart. Ultra-thin sections were stained with lead citrate and examined in a HT780 Hitachi electron microscope. Sample preparation and imaging were generated in the University of Valencia Electron Microscopy Service. Images were analyzed with ImageJ v.1.6.0.

Mitochondria isolation

Isolation of mitochondria from E18.5 tail tip—derived fibroblasts was performed from 1–3 150 mm plates, according to the differential centrifugation method. Briefly, cells were resuspended in medium A (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) and homogenized in a Teflon potter-type tissue homogenizer. The mixture was centrifuged at 1000 g for 5 minutes at 4 °C and the generated supernatant was subsequently centrifuged at 12000 g for 12 minutes at 4 °C. Finally, the pellet was resuspended in medium B (25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 0,05 mM EDTA, 5 mM MgCl₂, 10 mM Tris-HCl pH 7,4 and 10 mM H₃PO₄, final pH=7,4) and stored at −80 °C.

Mitochondria solubilization

Solubilization of mitochondrial membranes was carried out with digitonin in order to visualize both respiratory supercomplexes and free form complexes. The ratio of grams of detergent: grams of mitochondrial protein used was 4:1, except in gel where F₁F₀ATP synthase was visualized, in which the ratio was lowered to 2.5:1. 100 μg of purified mitochondria resuspended at 10 μg/μl (50 mM NaCl buffer, 50 mM imidazole and 5 mM aminocaproic acid) and containing digitonin at the desired concentration were incubated 10 minutes on ice. The insoluble portion was removed by centrifugation at 13000 g for 30 minutes at 4 °C. The pellet obtained was discarded and the supernatant was mixed with 4x loading buffer (5% Coomassie Blue-G250 in 1 M aminocaproic acid) for gel loading.

Blue Native-PAGE and Clear Native-PAGE

3–13% gradient polyacrylamide gels were prepared in house with a gradient former. Clear native (CN) gels were essentially the same as blue native (BN) gels, but 0.01% of digitonin was added to all gel solutions and Ponceau red buffer (Ponceau red, glycerol) was used instead of the normal loading buffer. The amount of sample loaded in each well was that obtained from solubilization of 100 μg of mitochondria. Cathode buffer A (tricine 50 mM, bis-tris 15 mM, pH 7.0, Coomassie Blue G-250 0.02%) and cathode buffer B (tricine 50 mM, bis-tris 15 mM, pH 7.0, Coomassie Blue G-250 0.002%) were used for electrophoresis. Electrophoresis was performed in a cold chamber. The first half hour was run at 90 volts with cathode buffer A. After that time, the cathode buffer was exchanged for cathode buffer B. Electrophoresis continued for approximately one more hour at 300 volts, until the dye began to run out of the bottom of the gel. In CN gels the whole electrophoresis was performed in cathode buffer B, with no changes.

In-gel complex I activity

Measurement of NADH dehydrogenase activity of complex I was determined on the same gel after BN-PAGE electrophoresis. The gel was incubated in 0.1 M Tris-HCl, pH 7.4, 0.14 mM NADH and 1 mg/ml NitroBlue tetrazolium solution at room temperature. Visualization was achieved by the purple precipitate produced after reduction of NitroBlue tetrazolium by the NADH dehydrogenase activity of complex I.

In-gel F₁F₀ATP synthase activity

Measurement of NADH dehydrogenase activity of F₁F₀ATP synthase was determined on the same gel after CN-PAGE electrophoresis. The gel was incubated in 270 mM glycine, 35 mMTris, pH 8.4 for 3 hours, then ATP hydrolysis was assayed in 270 mM glycine, 35 mMTris, 4 mM ATP, 14 mMMgSO4, 0.2%Pb(NO3)2, pH 8.4. The reaction was stopped after 5–10 min by 30 min incubation in 50% methanol/50% water after the formation of phosphate lead precipitates. The gel was then scanned.

Spectrophotometric activities

Measurement of complex I activity involved a two-step measurement, as described previously⁵⁶. First step was performed in 25 μg of 3x frozen-thawed mitochondria solubilized in Mg²⁺-containing C1/C2 buffer, DecylCoQ 130 μM and Antimycin A 1 μM. Absorbance measurement at 37 °C, 340 nm started after addition of NADH 100 μM and lasted 2–4 min. Then, rotenone 1 μM was added and absorbance was again measured for 2–4 min. Next, 1 mM Fe(CN)₆ was added and measurement was performed at 420 nm at 37° for 2–4 min. Later, Diphenyleneiodonium chloride (DPI) was added and absorbance was again captured in the same conditions. F₁F₀ATP synthase activity was measured as a readout of its ATPase activity. As previously reported⁵⁷, oligomycin-sensitive ATPase activity was calculated after measuring changes in absorbance at 340 nm driven by the pyruvate kinase reaction coupled to ADP phosphorylation by F₁F₀ATP synthase.

Analysis of mitochondrial membrane potential

For mitochondrial membrane potential imaging, E18.5 tail tip—derived wild-type and Tet3 knockout fibroblasts were seeded onto 96-well plates (PerkinElmer) and mitochondrial inhibitors were added at Krebs Ringer Phosphate Glucose buffer (KRPG) supplemented with NaCl 145 mM; Na₂HPO₄ 5,7 mM; KCl 4,86 mM; CaCl₂ 0,54 mM; MgSO₄ 1,22 mM; glucose 20 mM; pH 7,35. Next, cells were loaded with 10 nM TMRM (Sigma-Aldrich) and 1 µM cyclosporine-H (Sigma-Aldrich) in the Operetta CLS microscope (30 min at 37 °C in a 5 % CO₂ atmosphere) and confocal images were acquired at 40X, 1.4 NA objective (PerkinElmer). Mitochondrial uncoupler carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP, 10 µM, 15 min, Sigma-Aldrich) was added as a control of mitochondrial depolarization. Finally, images were analyzed using Harmony software (PerkinElmer).

Citrate synthase activities

E18.5 tail tip—derived fibroblasts from wild-type and Tet3 knockout embryos were snap-frozen. After three cycles of freeze/thawing, to ensure cellular disruption, citrate synthase activities were determined. Citrate synthase activity was measured in the presence of 93 mM Tris-HCl, 0.1% (vol/vol), Triton X-100, 0.2 mM acetyl-CoA and 0.2 mM DTNB; the reaction was started with 0.2 mM oxaloacetate, and the absorbance was recorded at 412 nm (30 °C) (e = 13.6 mM⁻¹·cm⁻¹)⁵⁸. The protein concentration of the samples was quantified by the BCA protein assay kit (Thermofisher) following the manufacturer’s instructions, using BSA as a standard.

Oxygen consumption measurement

Oxygen consumption assays were performed as previously reported⁵⁹. Briefly, oxygen consumption rate (OCR) was measured using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience). E18.5 tail tip—derived fibroblasts were plated 1 day before the experiment and preincubated with unbuffered DMEM 1 h at 37 °C in an incubator without CO₂ regulation. Successive injections of unbuffered DMEM, 5 μg/mL oligomycin, 300 nM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) and 1 μM rotenone plus 1 μM antimycin A were programmed. Calculations were performed following the manufacturer’s instructions.

Whole Genome Bisulfite Sequencing (WGBS) analysis

5mC levels and genomic location was determined using whole genome bisulfite sequencing on Tet3 knockout and wild-type E18.5 entire small intestinal epithelium. Two lines of each genotype were analyzed. Briefly, genomic DNA spiked with lambda DNA was fragmented to 200–400 bp. The DNA fragments were bisulfite treated using EZ DNA Methylation Gold kit (Zymo Research) to generate single strand DNA. Methylation sequencing adapters were ligated, followed by double strand DNA synthesis. Libraries were sequenced on an Illumina NovaSeq X Plus Series (PE150). Quality of the raw sequence reads was assessed with FastQC (fastqc_v0.11.8). Bismark software (version 0.24.0)⁶⁰ was used to perform alignments of bisulfite-treated reads to the reference genome. The reference genome was firstly transformed into a bisulfite-converted version and then indexed using bowtie2⁶¹. Sequence reads that produce a unique best alignment from the two alignment processes (original top and bottom strand) were then compared to the normal genomic sequence and the methylation state of all cytosine positions in the read was inferred. The same reads that aligned to the same regions of genome were regarded as duplicated ones. The sequences were divided into 10 kb bins to calculate their methylation level. The sum of methylated and unmethylated read counts in each window was calculated. Differentially methylated regions (DMRs) were identified using the DSS software⁶². According to the distribution of DMRs through the genome, genes were related to DMRs when their gene body region (from TSS to TES) or promoter region (upstream 2 kb from the TSS) have an overlap with the DMRs. Gene Ontology (GO) enrichment analysis of genes related to DMRs was implemented by the GOseq R package⁶³, in which gene length bias was corrected. GO terms with corrected p-value less than 0.05 were considered significantly enriched by DMR-related genes KOBAS software⁶⁴ was used to test the statistical enrichment of DMR- related genes in KEGG pathways. The WGBS and subsequent analysis were performed by Novogene. To identify putative enhancers on hypermethylated DMRs, H3K27ac and H3K4me1 ChIPseq data performed on murine intestinal cells at postnatal day 0 (P0) were downloaded from ENCODE (experiments: ENCSR642VYW and ENCSR159RVN) and used to identify enhancers in P0 intestinal cells (ENCFF660XDP.bed and ENCFF349IDR.bw for H3K27ac; ENCFF523POG.bed and ENCFF559NPS.bw for H3K4me1). Bedtools intersect was used to identify overlapping H3K27ac and H3K4me1 peaks with default parameters. Line plots and heatmaps were generated using deepTools.

MALDI-MSI measurement

All chemicals were purchased from Merck, if not otherwise mentioned. The first 2 cm of E18.5 mouse small intestines that include the whole duodenum and partial jejunum from wild-type and Tet3 knockout embryos were dissected and fresh frozen in liquid nitrogen. Then, the samples were embedded in 7.5%HPMC-2.5%PVP⁶⁵ and cryo-sectioned for MALDI-MS imaging as described elsewhere⁶⁶. Briefly, tissue sections were collected at 20 µm thickness and thaw mounted on Intellislides purchased from Bruker Daltonics. Slides were put in plastic mailers, vacuum sealed and stored at −80 °C until use. For MALDI MSI, a DAN-HCl matrix solution was prepared: 10 mg 1,5-diaminonaphtalene (DAN), in 5 ml of solvent: 60% acetonitrile (ACN) in milliQ water (VWR) and 60 mM HCl. The matrix was sprayed using the HTX-M5 sprayer (HTX Technologies) with the following parameters: nozzle temperature 65 °C, passes 6, flow rate 0.07 ml/min, velocity 1200 mm/min, track spacing 2 mm, pattern CC, pressure 10 psi, drying time 10 s, and nozzle height 40 mm. After spraying, red phosphorus suspended in acetone was spotted on the slide for mass calibration purposes. MALDI MSI data was acquired on a timsTOF fleX system (Bruker Daltonics) at 5 µm in negative ion mode using 1,5-diaminonaphtalene (DAN) as matrix. MALDI MSI data was acquired on a tims-TOF fleX system (Bruker Daltonics) equipped with a smartbeam 3D 10 kHz laser and microGRID, controlled by TimsControl v4.1 and flexImaging v7.2 software (Bruker Daltonics). Data was acquired in negative ion mode, m/z range of 80–1300, with 25 laser shots per pixel, 10 kHz laser frequency, laser spot size 5 µm² and raster 5 µm. The tune parameters were the following: MALDI Plate Offset 50 V, Deflection 1 Delta −70 V, Funnel 1 RF 180 Vpp, isCID Energy 0 eV, Funnel 2 RF 180 Vpp, Multipole RF 180 Vpp, Collision Energy 3 eV, Collision RF 800 Vpp, Quadrupole Ion Energy 5 eV and Low Mass 70 m/z, Focus Pre TOF Transfer Time 80 µs and Pre Pulse Storage 6 µs, and Detection set to Focus Mode. After MALDI measurement, the matrix was removed by dipping the slide for 2 min in 100% methanol. An automated staining apparatus, SunTissuePrep (SunChrome) was used for hematoxylin and eosin staining. A coverslip was mounted on top using Eukitt as quick-hardening mounting medium. Data was imported into SCiLS Lab MVS, Version 2024b Pro, from Bruker Daltonics, using the standard import parameters. High resolution scan of the H&E stained slide was imported as “.svs” file using the Import Optical Image tool. The overview images and the high resolution scan were co-registered using three anchor points. For untargeted analysis, a first feature selection was done by setting an intensity threshold of 30.000 a.u. using the Sliding Window tool. A list of 2926 features was selected and used for bisecting k-means clustering in order to automatically separate the tissue area from the background area. A second feature list was generated using the Find Discriminating Features (ROC) tool with the threshold set to 0.65. A new list of 829 features was used for further analysis. Bisecting k-means segmentation using the new feature list, weak de-noising and correlation distance metric generated four regions of interest: red = muscle, yellow = base of the villi, green = center of the villi and purple/blue = tip of the villi. The villi were manually annotated based on the H&E staining scan and the segmentation image. The base and tip of four villi from each condition (WT and KO) were selected for statistical analysis. Annotation of features was done using HMDB database with 10 ppm mass error tolerance, adduct and isotope pattern matching through MetaboScape API inside SCiLS. All data was normalized by root-mean-square (RMS).

Cell culture

E18.5 tail tip—derived fibroblasts were cultured on gelatin using the following culture media: Knockout DMEM, 10% bovine fetal serum, 1X penicillin/streptomycin, 1X non-essential amino acids, 2 mM Glutamine and 0,1 mM β-Mercaptoethanol. Atp5e rescue experiments were performed on fibroblasts derived from E13.5 embryos and cultured as described above. Tet3 knockout and wild-type fibroblasts were transduced with defective ecotropic retroviral particles coding for Atp5e or empty. The retroviral vector pMSCV-puro (Clontech) and the packaging pCL-Eco plasmid (Addgene #12371)⁶⁷ were used to generate the retroviral particles. After transduction, fibroblasts were selected with 1 μg/mL puromycin during 5 days. All experiments were performed using fibroblasts at passage 3.

Metabolomic analysis

Cells were fixed with 80% methanol, at 100 μL per 1 million cells. Cells were lysed with the aid of two metal balls at 30 Hz on a MM 400 mill mixer for 3 min, followed by centrifugal clarification at 21,000 g for 10 min. The clear supernatants were collected for the following assays. The precipitated pellets were used for protein assay using a standardized BCA procedure. For NAD and nucleotide quantification, an internal standard (IS) solution containing ¹³C or ²H-labeled NAD, NADH, AMP and ATP was prepared in 50% methanol. Serially diluted standard solutions of the targeted metabolites were prepared in 80% methanol. 10 μL of the clear supernatant of each sample or each standard solution was mixed with 40 μL of IS solution. 10 μL aliquots of the resultant solutions were injected into a C18 column to run UPLC-MRM/MS with (−) ion detection on a Waters Acquity UPLC system coupled to a Sciex QTRAP 6500 Plus MS instrument, using a tributylamine buffer solution and acetonitrile as the mobile phase for binary-solvent gradient elution. For GSH and GSSG quantification, an internal standard (IS) solution containing isotope-labeled GSH and GSSG was prepared in water. Serially diluted standard solutions of GSH and GSSG were prepared in 80% methanol. 20 μL of the clear supernatant of each sample or each standard solution was mixed with 80 μL of the IS solution. 10 μL aliquots of the resultant solutions were injected into a HILIC column to run UPLC-MRM/MS with (+) ion detection on an Agilent 1290 UHPLC system coupled to an Agilent 6495B QQQ MS instrument with the use of 0.1% formic acid in water and in acetonitrile as the mobile phase for binary-solvent gradient elution. For analysis of glucose, glucose-6P and mannose-6P, 50 μL of the supernatant of each sample was mixed with 50 μL of a solution of ¹³C6-glucose as internal standard, 100 μL of 25 mM AEC solution and 20 μL of acetic acid. The mixture was allowed to react at 60 °C for 70 min. After reaction, 300 µL of water and 300 µL of dichloroform was added. The mixture was vortex mixed and then centrifuged. The supernant was injected in 10-μL aliquots to run UPLC-MRM/MS with positive-ion detection on an Agilent 1290 UHPLC system coupled to a 4000 QTRAP mass spectrometer, as previously described⁶⁸. The analysis of the TCA cycle carboxylic acids was carried out as previously described⁶⁹. Briefly, 20 μL of the supernatant was mixed with 20 μL of a D- or 13-labeled analogue for each analyte as the internal standard, 20 μL of 200 mM 3-NPH solution and 20 μL of 150 EDC-6% pyridine solution. The mixture was allowed to react at 30 °C for 40 min. After reaction, the solution was diluted 3 fold with water. 20 μL was injected to run UPLC-MRM/MS with negative-ion detection on an Agilent 1290 UHPLC system coupled to a 4000 QTRAP mass spectrometer. Finally, quantification of other metabolites were performed as follows. 50 µL of each supernatant was mixed with 50 µL of water containing 50 pmol/mL GTP-13C10 and 50 µL of chloroform. The mixture was vortex mixed for 1 min, followed by centrifugation. 50 µL of each clear supernatant was taken out and mixed with an equal volume of water. 10 µL was injected to run UPLC-MRM/MS with negative-ion detection using a custom-developed reversed-phase LC-MRM/MS method for binary-solvent gradient elution. Concentrations of the detected analytes in the above assays were calculated with internal standard calibration by interpolating the constructed linear regression curves of individual compounds. The metabolomic analysis was performed in UVic Genome BC Proteomics Centre (Canada).

Statistical analysis

Data, unless otherwise stated, is represented as mean ± standard deviation. Two-tailed t-test was used to assess significant differences between groups.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-54044-0