Mouse studies
Wild-type C57BL/6 male mice (6–8 weeks of age) were purchased from BIKAI. Nlrx1−/− mice were purchased from Cyagen. NSG mice were purchased from Shanghai Model Organisms Center. All mice were housed in the specific-pathogen-free animal facility of Fudan University with the following environmental parameters: temperature maintained at 21–25 °C, relative humidity at 45–65% and a 12 h–12 h light–dark cycle.
All mice were randomly separated into each experiment group. Mice were fasted from 10:00 for 24 h with free access to water without food. PBS or HC (59847, Sigma-Aldrich; 100 mg per kg) was intraperitoneally injected into mice at 10:00 for 4 h. For acetate administration, mice were fasted for 24 h and PBS or sodium acetate (S5636, Sigma-Aldrich; 1 g per kg) was intraperitoneally injected 10 h and 1 h before mice were euthanized. For serum collection, mice were fasted overnight for 16 h with free access to water. For food reintroduction, mice were fasted for 24 h and then re-fed for another 24 h. Mice were euthanized and the indicated tissues were collected for subsequent analysis.
For the KPC model used in the MRTX1133 therapy experiment, 1 × 106 KPC cells were subcutaneously injected into 6- to 8-week-old NSG mice. The vernier calliper measurements begun when the tumours reached around 200 mm3. Tumour volume measurements were recorded three times per week using the formula 0.5 × length × width2. A blinded study design was used in the mouse tumour experiment to prevent bias during data collection and assessment, thus mice were randomized into control and treatment groups, and treated by intraperitoneal injection with vehicle (10% DMSO + 90% (20% SBE-β-CD in saline) or MRTX1133 in vehicle (30 mg per kg, twice a day) when the tumour volume reached around 300 mm3. Tumours were collected after 6 days of treatment. All of the animal experiment procedures, including the maximal tumour volume, were approved by ethics committee of Department of Laboratory Animals, Fudan University.
AAV production and infection in vivo
Plasmids for the AAV2/9 system, including pAAV RC2/9 plasmids, pAAV helper plasmids and transgene plasmids with the CMV or U6 promoter were used for global expression or the knockdown of genes in vivo respectively as previously described44. The plasmids were mixed with PEI solution and transfected into HEK293T cells. Then, 60–72 h after transfection, the cells and medium were collected by centrifugation (3,500 rpm, 4 °C, 5 min). 5× polyethylene glycol (40% PEG 8000, 2.5 M NaCl) was added to the supernatant and incubated at 4 °C overnight followed by centrifugation (3,000 rpm, 4 °C, 5 min) to collect the virus pellet. Meanwhile, the cell pellet was resuspended with lysis buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.0) and lysed by three freeze–thaw cycles between liquid N 2 and a 37 °C water bath followed by centrifugation (5,500 rpm, 4 °C, 10 min) to obtain the supernatant. Then the supernatant was mixed with the virus pellet. The mixture was purified by Optiprep (D1556-250mL, Sigma-Aldrich) gradients (17%, 25%, 40% and 60%) centrifugation (40,000 rpm, 16 °C, 2 h). The viral fraction was collected from the 40% gradient, then washed three times with PBS using 100 kDa columns (3,500 rpm, 4 °C, 30 min).
AAVs were administered to C57BL/6J mice through gastrocnemius injection (5 × 1010 copies, 25 μl per mouse, three sites) or tail injection (1 × 1011 copies, 150 μl per mouse). All experiments were performed 3–4 weeks after AAV injection. The efficiency of Nlrx1 knockdown or overexpression mediated by AAV delivery was validated by immunoblotting.
Plasmids, reagents and antibodies
Plasmids
WT NLRX1 (HA tag), the NACHT domain (amino acids 160–483, HA tag), LRR domain (amino acids 669–975, Flag tag), ΔLRR (amino acids 1–668, HA tag), 4A (four sites, Glu729, Lys754, Gln758 and Arg958, were mutated to Ala, HA tag) of NLRX1, NLRX1-GFP11 (the C terminus of NLRX1 without stop codon was fused to the linker GGSGGGS and the GFP11 tag RDHMVLHEYVNAAGIT), NLRX1-GFP11-IRES-RFP (the C terminus of NLRX1-GFP11 fused to IRES and RFP), HSP60-GFP11 (the C terminus of HSP60 without stop codon was fused to the linker and the GFP11 tag), GFP11-TOM20 (the GFP11 and the linker fused to the N terminus of TOM20) and HA-NLRP3 were constructed into pcDNA3.1 vector; WT NLRX1-HA, ΔLIR-NLRX1 (amino acid deletion 461–466, HA tag), NLRX1(ΔN-ter) (amino acids 156–975, HA tag), NLRX1(Cyto) (amino acids 87–975, Flag tag), NLRX1(ER) (the N terminus of NLRX1(Cyto) fused to the amino acids 81–250 of FAM134B, Flag tag) were generated as previously described6; cytoGFP(1–10), matrixGFP(1–10) (the N terminus of GFP1–10 was fused to the MTS of COX8, residues 1–36) and GFP-Parkin were generated into the pLVX-hygro vector, and GFP-LC3B was generated into pQCXIH. Constructs encoding mt-Keima and MTS-eGFP were generated into the pLVX or pLVX-Tet-On vector.
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