L leu

The following materials are used in the cell culture: L-leu (#L8912), lysine (Lys, #L9037), arginine (Arg, #A6969), Collagen Ⅰ (#SCR103), Basic Fibroblast Growth Factor (bFGF, #13256-029), BSA (A1933), and DMSO (#C6295), which were purchased from Sigma-Aldrich, USA. DMEM (#11995065), L-leu-deprived DMEM powder (#88425), fetal bovine serum (FBS, #10099141C), Antibiotics-antimycotic (#15240062), and Dulbecco phosphate-buffered saline (DPBS, #14190144) were purchased from Gibco, Life, Technologies, Grand Island, NY, USA. Culture dish (10 and 6 cm), Cell ware 6, 24, and 96-well plates, Matrigel and cell strainer, were purchased from Corning, NY, USA. Accutase, Cell Detachment Solution was purchased from innovative cell technologies, SD, USA. Mouse monoclonal anti-PAX7 (#sc-81648) was purchased from Santa Cruz Biotechnology, CA, USA. Donkey anti-Mouse IgG (H + L) was purchased from Invitrogen (#A10036, Invitrogen, Carlsbad, CA, USA). DAPI (#C0065), Triton X-100 (#9002-93-1), and Tween 20 (#9008-64-5) were purchased from Solarbio (Beijing, China). CCK 8 was purchased from Good Laboratory Practice Bioscience, Montclair, CA, USA.

L-trp and L-leu (>97% pure) were purchased from Sigma Aldrich Chemical Company, Schnelldorf, Germany. Glucose was purchased from Hänseler, Herisau, Switzerland. ¹³C-sodium acetate was purchased from ReseaChem, Burgdorf, Switzerland.

2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (5 mg/mL, Sigma) was used to label the zebrafish intestines at 7 dpf. Embryos were treated with DCFH-DA in 0.3× Danieau buffer for 2 h. PED6 (D23739, Thermo Fisher) and EnzChek (E6639, Thermo Fisher) were used to test the digestive ability of the intestinal proteins and lipids. Embryos at 7 dpf were treated with 3 μg/mL PED6 or 20 μg/mL EnzChek in a 0.3× Danieau buffer for 3 h. rapamycin was used to inhibit the mTORC1 pathway. Embryos were exposed to 400 and 800 nM rapamycin (Sangon Biotech, China) in a 0.3× Danieau buffer from 10 hours postfertilization (hpf) to 3 dpf. L-Leu and rheb mRNA were used to elevate the mTORC1 pathway. Embryos were injected with L-Leu (500 nM, Sigma) at 30 hpf or rheb mRNA (100 pg and 150 pg) at 1-cell stage, then harvested at 3 dpf.

L-Leu (purity ≥ 98.5-101.0%), KIC (purity ≥ 98 %), and HMB free acid (purity ≥ 95%) were purchased from Sigma (St. Louis, MO, USA). TRIzol, DNase I, and SYBR Green detection kit were purchased from Invitrogen (Life Technologies, Carlsbad, CA, USA). Protease inhibitor cocktail was purchased from Roche (Basel, Switzerland). Phosphatase inhibitors were purchased from Thermo Scientific (Waltham, MA, USA). Phosphate Buffered Saline (PBS) and Trypsin were also purchased from Wisent. Mesotrione (2-(4-Mesyl-2-nitrobenzoyl)-1,3-cyclohexanedione)-Pestanal©, catalogue No. 33855) was obtained from Fluka (St. Louis, MO, USA). The growth medium used for cell growth consisted of high glucose Dulbecco’s modified Eagle’s medium (DMEM) purchased from Gibco (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (FBS) (Gibco #26050-088), and 1% Antibiotic-Antimycotic (Wisent #450-115-EL). The medium used for differentiation of cells was high glucose DMEM supplemented with 2% horse serum (HS) (Gibco #26050088) and 1% Antibiotic-Antimycotic.

The rTaq DNA polymerase, Takara MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0, ss-DNA, and RNase A were provided by Takara Bio Inc. (Tokyo, Japan). A yeast DNA Kit used was the product of Omega Bio-tek Inc. (Feyou Biotechnology, Shanghai, China). Lithium acetate dehydrate was purchased from Guangzhou Jinhauda Chemical Co., Ltd. (Guangzhou, China). Polyethylene glycol (MW4000), cetyl-tri-methyl-ammonium bromide (CTAB), Tris-HCl, sodium acetate, ethylene-di-amine-tetra-acetic acid (EDTA), ethidium bromide (EB), and agar were obtained from Sanli Chemical Company (Yangling, China). Bacto-yeast extract, bacto-peptone, and dextrose were purchased from Beijing Aoboxing Bio-tech Co. Ltd. Analytically pure L-Leu, L-Ile, standard hexaldehyde, standard isoamyl alcohol, standard 3-methylbutyl acetate, G4l8, and ampicillin were supplied by Sigma-Aldrich Corporation (Beijing, China).

Cell viability was evaluated by MTS assay as described.^{43 (link)} Jurkat cells (5×10⁴/well) were seeded into a 96-well plate in quadruplicate in both the absence and presence of the inhibitors Z-VAD-fmk (10 μM) (Bachem, Bubendorf, Switzerland) or Kp7-6 (0.1–0.5 mM) (Calbiochem, Billerica, MA, USA) in the culture medium. After an appropriate time of incubation, cells were treated with either ApLAO or CgLAO at different concentrations (0.25–10 μg/ml) for 12–48 h). When required, catalase (1000 U/ml) (Sigma) and/or L-Leu (790 μg/ml) (Sigma) were added to the culture medium containing ApLAO or CgLAO. MCF7 cells (2×10⁴/well) were seeded into a 96-well plate in quadruplicate. The next day, cells were treated as described above for Jurkat cells. Cell viability was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), in accordance with the manufacturer’s instructions. Absorbance was measured with an automatic microplate reader (Tecan Safire2; Tecan Group Ltd, Männendorf, Switzerland) at a wavelength of 492 nm. Results are presented as percentages of the corresponding vehicle treatment (control cells).