Chloroform

RT-qPCR analysis were performed as described previously^{101 (link)} with minor modification. Total RNA was isolated from purified or cultured cells by using Sepasol (Nacalai Tesque) and chloroform (Nacalai Tesque), precipitated by using 2-propanol (Nacalai Tesque), and washed with 75% (vol/vol) ethanol (Nacalai Tesque). RNA samples were incubated with DNase I (Invitrogen, Carlsbad, California, USA) to remove contaminating genomic DNA and then reverse-transcribed into cDNA (Superscript III reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Quantitative PCR analysis was performed by using a LightCycler 480 II (Roche, Basel, Switzerland) with FastStart Essential DNA Probes Master (Roche) or SYBR Green I Master reagents (Roche). Primer sequences were: Tnfα sense, 5′-CTGTAGCCCACGTCGTAGC-3′; Tnfα anti-sense, 5′-TTGAGATCCATGCCGTTG-3′; S100a8 sense, 5′-TCCTTGCGATGGTGATAAAA-3′; S100a8 anti-sense, 5′-GGCCAGAAGCTCTGCTACTC-3′; Pparγ sense, 5′-GAAAGACAACGGACAAATCACC-3′; Pparγ anti-sense, 5′-GGGGGTGATATGTTTGAACTTG-3′; Nrf1 sense, 5′-GCTCTCTGAGACGCTGCTTT-3′; Nrf1 anti-sense, 5′-GTGTTCAGTTTGGGTCACTCC-3′; Actb sense, 5′-AAGGCCAACCGTGAAAAGAT-3′; and Actb anti-sense, 5′-GTGGTACGACCAGAGGCATAC-3′.

Metabolite extraction from lung tissue for comprehensive metabolomic profiling was performed using the slightly modified protocol of a previously established method^{56 (link)}. Briefly, frozen tissue samples were combined with a 500-μL aliquot of ice-cold methanol containing methionine sulfone (L-Met) and 2-morpholinoethanesulfonic acid (MES), serving as designated internal standards (IS) for cationic and anionic metabolites, respectively. This mixture was subjected to homogenization using a Finger Masher manual homogenizer (AM79330, Sarstedt, Tokyo, Japan). To this homogenate, half the volume of ultrapure water (LC/MS grade, obtained from Wako) and 0.4 times the initial volume of chloroform (Nacalai Tesque, Kyoto, Japan) were added. The resulting mixture was centrifuged at 15,000 × g for 90 min at a temperature of 4 °C. After centrifugation, the resulting aqueous phase was subjected to filtration using an ultrafiltration tube (Ultrafree MC-PLHCC, Human Metabolome Technologies, Yamagata, Japan). The filtrate was then concentrated by nitrogen flow-assisted evaporation on a heating block (DTU-28N, TAITEC, Koshigaya City, Japan). The concentrated filtrate was resuspended in 50 μL ultrapure water for subsequent LC-MS/MS and IC-HR-MS analytical procedures.

α-N-Hydroxysuccinimidyl-ω-Boc protected-amino-PEG (NHS-PEG-NH₂-Boc, Mw: 5000), 1,2-dimyristoyl-sn-glycerol-3-phosphatidylethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE), and 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine (DSPE) were purchased from NOF Corporation (Tokyo, Japan). Dichloromethane, chloroform, toluene, diethyl ether, and n-butyl amine were obtained from Nacalai Tesque (Kyoto, Japan). Methoxyl-PEG-succinimidyl propionate (MeO-PEG-NHS, Mw 5000) was purchased from Nektar Therapeutics (San Carlos, CA, USA). L-α-Phosphatidylcholine from egg yolk (EggPC) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Fluorescein isothiocyanate (FITC) was obtained from Dojindo Laboratories (Kumamoto, Japan). Phosphate-buffered saline (PBS) was obtained from Nissui Pharmaceutical, Co., Ltd (Tokyo, Japan). 1-Hexadecanethiol was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). Trifluoroacetic acid (TFA) and phospholipid C-test wako were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Octadecyl triethoxysilane was supplied from Shin-Etsu Chemical Co., Ltd (Tokyo, Japan). L-α-Palmitoyl-NBD dodecanoyl phosphatidylethanolamine was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA).

Reverse transcription and quantitative PCR analysis were performed as described previously [30 (link)]. In brief, RNA from cell suspensions was isolated by using Sepazol (Nacalai Tesque) and chloroform (Nacalai Tesque). After precipitation with 2-propanol (Nacalai Tesque) and washing with 75% (vol/vol) ethanol (Nacalai Tesque), the residue was incubated with DNaseI (Thermo Fisher Scientific) and reverse-transcribed to cDNA (Superscript 3 reverse transcriptase, VIRO cDNA Synthesis Kit; Invitrogen).
Real-time PCR was performed by using the LightCycler 480 System II (Roche, Basel, Switzerland) and SYBR Green I Master reagents (Roche). Primer sequences were: 5′-ggggatggagaagctacagg-3′ (sense) and 5′-tccgcttcaaacagagtgc-3′ (anti-sense) for Alox15, 5′-gaaagacaacggacaaatcacc-3′ (sense) and 5′-gggggtgatatgtttgaacttg-3′ (anti-sense) for Pparg and 5′-aaggccaaccgtgaaaagat-3′ (sense) and 5′-gtggtacgaccagaggcatac-3′ (anti-sense) for Actb.

Methoxyamine hydrochloride (quality level: MQ 200) was purchased from Merck (Darmstadt, Germany). Pyridine (extra dry) (99.5+% evaluated by capillary GC), acetone (Dioxins Analysis Grade, purity 99.8%), and d₁₀-phenanthrene (Environment Analysis Grade, purity 99.5%) were obtained from Wako Chemicals Ltd. (Osaka, Japan). The metabolite mixture kit for GC/MS-based metabolomics and N-methyl-N-(trimethylsilyl trifluoroacetamide) (MSTFA) were purchased from GL Sciences Inc. (Tokyo, Japan). Acetonitrile (LC-MS grade) and methanol (LC-MS grade) were purchased from Kanto Chemical (Tokyo, Japan). Standard Reference Material (SRM) 1950 “Metabolites in Human Plasma” was obtained from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). Adipic acid (purity 99.5%), chloroform (purity 99.0%), and methanol (purity 99.8%) were obtained from Nacalai Tesque Inc. (Kyoto, Japan).

Total RNA was extracted from the cortex using the Tri reagent (Molecular Research Center, Cincinnati, OH, USA). After euthanasia, we rapidly sampled the cortex of 24-week-old KCASP1Tg mice (n = 5) and WT mice (n = 5). RNA was isolated using chloroform (Nacalai Tesque, Kyoto, Japan) and precipitated using isopropanol (Nacalai Tesque). The RNA concentration was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Worsham, MA, USA). Extracted RNA was transported to KURABO INDUSTRIES LTD (Osaka, Japan) for microarray analysis. The results were analyzed using ”Transcriptome Viewer”. Heat maps and clustering of significant miRNAs are presented for the microarray experiments.