Mouse studies
Male C57BL/6 mice, aged 6 to 8 weeks, were sourced from BIKAI, while Nlrx1−/− mice came from Cyagen, and NSG mice were obtained from the Shanghai Model Organisms Center. All mice lived in a specific-pathogen-free environment at Fudan University, where conditions included a temperature between 21–25 °C, humidity of 45–65%, and a 12-hour light-dark cycle.
The mice were assigned randomly to various experimental groups. A fasting protocol was implemented, where mice had no food but could drink water for 24 hours starting from 10:00 AM. They received either PBS or HC (59847, Sigma-Aldrich; 100 mg per kg) through intraperitoneal injection at 10:00 AM for a 4-hour duration. For acetate administration, after a 24-hour fast, they were administered PBS or sodium acetate (S5636, Sigma-Aldrich; 1 g per kg) via intraperitoneal injection at intervals of 10 and 1 hours before euthanasia. Blood samples were collected after a 16-hour overnight fast with access to water. After a 24-hour fasting, mice were re-fed for an additional 24 hours before being euthanized to collect specific tissues for analysis.
In the KPC model for the MRTX1133 therapy experiment, 1 × 106 KPC cells were injected subcutaneously into NSG mice aged 6 to 8 weeks. Tumour measurements started when they reached approximately 200 mm3. Recordings of tumour volumes were taken three times weekly using the formula 0.5 × length × width2. The experimental design for the tumour study was blinded to avoid bias, with random assignment into control and treatment groups. Treatment involved intraperitoneal injections of either a vehicle (10% DMSO + 90% (20% SBE-β-CD in saline)) or MRTX1133 in the vehicle (30 mg per kg, twice daily) once tumours reached about 300 mm3. Tumour specimens were harvested after 6 days of treatment. All animal procedures, including the maximum allowed tumour size, received approval from the Fudan University ethics committee for Laboratory Animals.
AAV production and infection in vivo
Plasmids for the AAV2/9 system, which included pAAV RC2/9 plasmids, helper plasmids, and transgene plasmids with either CMV or U6 promoters, were utilized to express or knock down genes in vivo, as previously documented. The plasmids were combined with PEI solution before being transfected into HEK293T cells. After 60 to 72 hours post-transfection, both the cells and culture media were collected via centrifugation (3,500 rpm, 4 °C, 5 minutes). A fivefold polyethylene glycol solution (40% PEG 8000, 2.5 M NaCl) was added to the supernatant and allowed to incubate at 4 °C overnight, followed by another round of centrifugation (3,000 rpm, 4 °C, 5 minutes) to retrieve virus pellets. Meanwhile, cell pellets were resuspended in lysis buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.0) and lysed using three freeze-thaw cycles with liquid nitrogen followed by a 37 °C water bath, then centrifuged (5,500 rpm, 4 °C, 10 minutes) to obtain the supernatant. This was then mixed with the virus pellet and purified via Optiprep (D1556-250mL, Sigma-Aldrich) gradient centrifugation (17%, 25%, 40%, and 60%) at 40,000 rpm for 2 hours at 16 °C. The viral fraction was collected from the 40% gradient and washed three times with PBS using 100 kDa columns (3,500 rpm, 4 °C, 30 minutes).
AAVs were administered to C57BL/6J mice either through gastrocnemius injection (5×1010 copies, 25 μl per mouse, three sites) or via tail injection (1×1011 copies, 150 μl per mouse). All assessments were taken 3 to 4 weeks following AAV injection, with the effectiveness of Nlrx1 knockdown or overexpression verified through immunoblotting.
Plasmids, reagents and antibodies
Plasmids
WT NLRX1 (with HA tag), various NLRX1 domains (such as NACHT and LRR with respective tags), plus constructs for NLRX1-GFP11 and others were prepared in the pcDNA3.1 vector. Other plasmids, including cytoGFP(1–10), were introduced into pLVX-hygro, and several guide RNAs targeting NLRX1 were designed online and inserted into the pLentiCRISPR v2 vector for gene editing purposes.
For NLRX1–HA knock-in cell creation, a specific guide RNA was cloned into the pX458 plasmid. Homology arms for the NLRX1 sequence were also introduced into the pcDNA 3.1 vector, adjusted with a mutation prior to the HA tag insertion.
Metabolites
Metabolites like AcCoA sodium salt, CoASH, malonyl-CoA, and sodium acetate were obtained from Sigma-Aldrich, while succinyl-CoA was sourced from MCE. Biotin was linked to amino groups of AcCoA per the manufacturer’s directions.
Antibodies
A variety of antibodies were employed in immunoblotting, including those against TIM23, LC3, HSP60, and others. Fluorescent secondary antibodies were used for immunofluorescence experiments.
Inhibitors
Several inhibitors, including HC, BTC, and CCCP, were acquired from various suppliers for experimentation.
Cell culture and cell line generation
Cell lines such as HEK293T, HeLa, A549, MCF7, and U-2 OS were sourced from ATCC, with additional lines from NCACC. Sf9 cells were obtained from Invitrogen. The KPC cell models were provided by Z.-G. Zhang. Cells were generally cultured in DMEM or RPMI-1640 media supplemented with FBS and a penicillin-streptomycin mixture. Importantly, each cell line underwent testing to ensure they were free from mycoplasma contamination, whereas specific conditions applied for Sf9 cells.
CRISPR–Cas9 was utilized to generate NLRX1-knockout cells. Selected cells were validated through sequencing and immunoblotting to confirm the knock-out status. Similar methods were applied for generating NLRX1–HA-tag knock-in cells.
To express mt-Keima in an inducible manner, HeLa cells underwent specific infection protocols and selection strategies before being confirmed for the desired expression via various means.
Virus packing
Lentiviral or retroviral vectors containing the specified genes were transfected into HEK293T cells alongside packaging plasmids. After a set period, collected supernatants underwent filtering and concentration.
Gene knockdown by siRNA
SiRNAs were introduced into cells through Lipofectamine RNAiMAX application per the manufacturer’s guidelines, and subsequent treatments were applied after 48 hours before analysis.
Mitochondria isolation
Mitochondria were isolated following a specific lysis buffer protocol. This included washing cells with cold PBS, applying mitochondrial lysis buffer, and using sequential centrifugation steps to separate cytosolic and mitochondrial fractions.
Immunoblotting and related assays
Various techniques were employed for immunoblotting, including lysing cells with SDS buffer, followed by analyses using SDS–PAGE. Specific optional protocols were also outlined for various assays and controls involved in the research.
Purification of recombinant NLRX1 proteins
Human NLRX1 was cloned with specific tags and subjected to purification processes involving bacterial transformation and chromatography techniques to confirm successful yields for further assays.
[3H]AcCoA binding assay
The binding assay was executed with recombinant proteins using dextrin beads and analyzed through various washing and incubation steps, followed by quantification using a scintillation counter.
Protein oligomerization analysis
This analysis involved cross-linking samples to assess oligomerization through specific protocols followed by electrophoresis to determine protein interactions.
Molecular modelling for AcCoA binding to NLRX1
Protein structures were sourced from the Protein Data Bank and simulations were carried out to explore binding interactions. Visualization of the structures was performed using Pymol software.
Flow cytometry
Cell treatments preceded flow cytometry for analyzing mitochondrial dynamics and localization of proteins via various fluorescence channels and settings.
ATP measurement and cell death assay
Intracellular ATP production was measured through a luminescence-based assay, while cell death was assessed using a non-radioactive cytotoxicity assay kit, following prescribed methodologies.
Measurement of NADP(H)
Utilizing a specific assay kit, NADP+ and NADPH were measured through dilution and incubation assays to ascertain levels within cell samples.
2D cell proliferation
Cells underwent treatment with serially diluted compounds to analyze growth, with luminescent detection performed to calculate cell viability and response variables.
Metabolite extraction and GC–MS
For studies on intracellular metabolites, specific cells were subject to detailed extraction processes, followed by gas chromatography and mass spectrometry for analysis of various metabolites.
Metabolite extraction and LC–MS
AcCoA levels were measured using established protocols for cytosolic and mitochondrial fractions, with various analytical steps ensuring detailed analysis of the biochemical environment.
Seahorse analysis
The mitochondrial oxygen consumption rate was measured in specific cell lines using the Seahorse equipment to assess cellular respiration under various treatment conditions.
CRISPR screening
For generating lentivirus for screening, a genome-wide library was utilized, allowing for large-scale screenings with specific follow-up validations through sequencing analyses.
qPCR analysis
Genomic DNA was isolated from cells with qPCR performed to amplify specific mitochondrial and nuclear genomic targets, employing full validation procedures to ensure accuracy.
Immunofluorescence and confocal microscopy
Cells underwent specific treatment protocols and were prepared for confocal microscopy to assess the localization and interaction of various proteins, ensuring robust imaging practices.
Mitophagy reporter assay
The assay involved both in vivo and in vitro methods of evaluating mitophagy levels in mice and cell cultures, utilizing high-throughput analysis through various detection techniques.
Cytochrome c release analysis
HeLa cells were treated and lysed for analysis of cytochrome c release into the cytosol, employing multiple centrifugation steps for clarity in results.
Tissue mitochondrial isolation
Protocols were established for isolating mitochondrial fractions from mouse tissues, with minimizing contamination and ensuring valid sample processing throughout.
Electron microscopy
Cells were treated, collected as suspension, and subjected to fixation for electron microscopy, providing insights into cellular structures at nano-scale levels.
Thermal shift assay
Examination of proteins involved heating and centrifugation steps for solubility assessments, leading to further immunoblotting for validation.
Statistical analysis
Data analyses used GraphPad Prism or Excel, including detailed statistical summations and a variety of methods to ensure reliability and accuracy of the presented data.
Reporting summary
Further details on the research design can be found in the linked reporting summary.
