Tricaprylin

E. coli BL21 (DE3), pET28a (+) (Novagen, WI, USA), pT7–7 (USB, OH, USA) and IPTG (Isopropyl β-D thiogalactopyranoside) (Invitrogen Life Technologies, CA, USA) were used in the recombinant protein expression system. The HiTrap Chelating Sepharose HP column (HiTrap) was purchased from GE Healthcare (Uppsala, Sweden). Activity assays involved the substrates triolein (C18, purities of 65% and 99%), tricaprylin (C8, 99%), caprylic acid (C8:0, 99%), lauric acid (C12:0, 99%), myristic acid (C14:0, 99%), palmitic acid (C16:0, 99%), stearic acid (C18:0, 99%), oleic acid (C18:1, 90%) and linoleic acid (C18:2, 99%) (Sigma-Aldrich, St. Louis, MO, USA). For the immobilization, polypropylene beads (Accurel MP 1000, Membrane GmbH, BY, Germany) were used, with the following characteristics: surface area of 55.985 m² g^-1, particle density of 1.993 g cm^-3 and particle diameter <1500 mm. All other reagents and substrates used were of analytical grade.

Acylglycerols of at least 95% purity were used in this study. Tricaprylin (purity > 99%) was purchased from Sigma Aldrich (St. Louis, MO, USA). 1-monocaprylin (> 98%), 1-monocaprin (> 98%), 1-monolaurin (> 98%), tricaprin (> 98%), 1,3-dilaurin ( > 96%), trilaurin (> 98%), and trilinolein (> 95%) were obtained from TCI Europe (Zwijndrecht, Belgium). Triolein (> 95%) was purchased from Fisher Scientific (Waltham, MA, USA). The structure and properties of the acylglycerols are given in Table S1. A buffer consisting of 2 mM Tris-maleate, 1.4 mM CaCl₂ and 150 mM NaCl with a pH of 6.5 was used in this study and is referred to as “lipolysis buffer.” All buffer components were purchased from Sigma Aldrich (St. Louis, MO, USA) and were of analytical grade. The medium used for in vitro lipolysis consisted of lipolysis buffer supplemented with fasted state simulated intestinal fluid (FaSSIF) instant powder (biorelevant.com, London, UK); FaSSIF instant powder comprises the bile salt sodium taurocholate and lecithin. The concentration of sodium taurocholate and phospholipid in the lipolysis medium was 6 and 1.5 mM, respectively.

Single cells suspensions from spleen and bone marrow of femurs were prepared. Spleens were carefully passed through a 40 μm cell strainer (BD Biosciences, Heidelberg, Germany), washed twice with PBS/2% FCS and erythrocytes were lysed with hypertonic solution (0.17 M NH4Cl with 20 mM HEPES in H₂O) for 10 min at 37°C. For isolation of bone marrow cells epicondyles were cut and diaphyses were flushed with 5 ml 1x PBS. Cells were counted with a haemocytometer (A.Hartenstein, Würzburg, Germany). Peripheral blood for FACS-staining was isolated with EDTA-coated microvettes (Sarstedt, Nümbrecht, Germany).
The BFS1 fibrosarcoma cells were generated in a female C57BL/6N mouse by injection of 1 mg of 3-methylcholanthrene (Sigma, Taufkirchen, Germany) dissolved in 200 μl tricaprylin (Sigma) i.d. in the back of a mouse as described [35 (link)]. Syngeneic tumor cells (BFS1, 2x10⁵ in 50μl PBS) were subcutaneously implanted on the upper part of the loin. The diameters of the tumors were measured in two directions perpendicularly to each other with a caliper.

Escherichia coli (E. coli) DH5α (Invitrogen, Shanghai, China) and plasmid pET22b (Novagen, Madison, USA) were used as cloning host and vector, respectively. E. coli Rosetta-gami B (Weidi, Shanghai, China) was used for protein expression. PrimerSTAR Max DNA polymerase and the restriction endonucleases EcoRI, XhoI and DpnI were purchased from Takara (Dalian, China). Isopropyl β-_D-1-thiogalactopyranoside (IPTG), BSA standard protein and antibiotic ampicillin were purchased from Sangon (Shanghai, China). Triolein and tricaprylin were purchased from Sigma-Aldrich (Shanghai, China), and trilaurin was purchased from TCI (Shanghai, China). Other reagents were of analytical grade and provided by a local supplier.

Both RHT premade oral solution and RHF herbal mixture (composed of jack-in-the-pulpit tuber 9 g, common selfheal fruit-spike 12 g, seaweed 9 g, catclaw buttercup root 15 g, amorphophallus konjac 15 g, appendiculate cremastra pseudobulb 9 g, snakegourd peel 15 g, and dried tangerine peel 9 g, for each recipe) were obtained from the department of pharmacy, Longhua Hospital, affiliated to the Shanghai University of TCM, Shanghai, China. Benzo(a)pyrene (BP) and tricaprylin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Eight-week-old A/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA), fed, and maintained in the SIRF-West animal center at Washington University. The study was approved by Washington University’s Institutional Animal Care and Use Committee.

Male gpt delta mice (9-weeks old; body weight, ~25 g), which carry approximately 80 copies of lambda EG10 DNA on each chromosome 17 on a C57BL/6 J background [12 (link)], were obtained from Japan SLC (Shizuoka, Japan). The mice were maintained at 24 to 26 °C with 55 % to 75 % humidity and a 12-h light–dark cycle, and were fed CA-1 diet (Japan Clea Co., Tokyo, Japan) with water ad libitum, in the specific-pathogen–free (SPF) animal facility of the National Institute for Environmental Studies. The animals were anesthetized with 4 % halothane (Hoechst Japan, Tokyo, Japan) in a desiccator until they did not respond to a tactile stimulus. A single dose of B[a]P (1 mg, Wako Pure Chemical Industries, Osaka, Japan) dissolved in 50 μL of tricaprylin (Sigma-Aldrich, St. Louis, MO, USA) was intratracheally instilled via a polyethylene tube [11 (link), 13 (link)]. Control mice were treated with 50 μL of vehicle (tricaprylin) alone; this dose of B[a]P is within the range (0.5–2 mg) that causes a linear dose-dependent increase in mutant frequency in the lungs of 3-month–old gpt delta mice [11 (link)]. The mice were sacrificed 14 days after the administration, and their lungs were removed, frozen in liquid nitrogen, and stored at −80 °C until the DNA was isolated. The animal studies were approved by the Animal Care and Use Committee of National Institute for Environmental Studies.

B[a]P and tricaprylin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Chromium Single Cell 3′ v3 Reagent Kits (10x Genomics) and NextSeq 500/550 High Output sequencing reagent Kits v2 (150 cycles) (Illumina, San Diego, CA, USA) were used according to the manufacturer’s protocol [9 (link),10 (link),11 (link)]. LKR13 cells, a mouse lung adenocarcinoma line that expresses mutant KrasG12D on the SV129 background, were a generous gift from Dr. Jonathan M. Kurie (MD Anderson Cancer Center, Houston, TX, USA). LKR13-Luc cells were generated by transfecting LKR13 cells with a lentivirus expressing firefly luciferase (PLV-10064-200; Cellomics Technology, LLC, Halethrope, MD, USA). H2030-BrM3 cells were a generous gift from Dr. Joan Massage (Memorial Sloan Kettering Cancer Center, New York, NY, USA). All cell lines were grown in RPMI (Roswell Park Memorial Institute) 1640 medium (Cat# 11875; Thermo Fisher Scientific, Wiltham, MA, USA) supplemented with 10% fetal bovine serum at 37 °C in 5% carbon dioxide. A/J mice, FVB mice, and SV129 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mito-HNK and Mito-LND were synthesized as previously described [4 (link),8 (link)] (Figure 1A).