Glutamine

Ethylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood samples were collected from patients before surgery and healthy controls. Genomic DNA was extracted using a RelaxGene Blood DNA System (TIANGEN biotech, Beijing, China) according to the manufacturer's instruction. The fresh surgical specimens of GBM from 11 patients were harvested and immediately immersed in ice-cold PBS containing 1% penicillin/streptomycin and 0.5% glutamine (Beyotime, Shanghai, China). Primary cell culture was performed within 60 min after surgery. As a variation of phenotype might occur at higher passages, we chose the cell cultures at the fourth passage for functional experiments.

Intestinal epithelial cells (IEC-6) were cultured in DMEM (Invitrogen, Waltham, MA, USA) supplemented with 10% of fetal bovine serum (HyClone, Logan, UT, USA), 1% glutamine (Beyotime, Shanghai, China), and 1% penicillin-streptomycin (Beyotime, Shanghai, China) in a 5% CO₂ humidified incubator at 37 °C. The hypoxia/reoxygenation (H/R) model in vitro was established as previously described [25 (link)]. To simulate hypoxic conditions, IEC-6 cells were incubated in a microaerophilic system (Thermo, Waltham, MA, USA) with 5% CO₂ and 1% O₂ and balanced with 94% N₂ for 4 h. Then, IEC-6 cells were cultured for 3 h under normoxic conditions to achieve reoxygenation. Cells from the 3rd to 6th passages were used for the following experiments. When the cells grew to 80–90% confluence, they were pretreated with PTE (0.5 or 1 μM) (HPLC purity ≥ 98%, Must Bio-Technology, Chengdu, China) or DMSO (as the control; Sigma, Cleveland, OH, USA) at the indicated concentrations for 24 h before being exposed to H/R. To study the involvements of SIRT3 in the PTE-mediated protective effect against H/R, the selective SIRT3 inhibitor 3-TYP (MCE, Wuhan, China) and SIRT3 siRNA (Thermo Fisher, Waltham, MA, USA) were used to treat IEC-6 cells before PTE treatment.

TXYF was prepared with Rhizoma Atractylodis Macrocephalae, Radix Paeoniae Alba, Pericarpium Citri Reticulatae, and Radix Saposhnikoviae, which were purchased from the Wuxi Hospital of Traditional Chinese Medicine and composed in 6 : 4 : 3 : 2 proportions. Raw components were soaked in a 10 times volume of distilled water for 0.5 h and boiled twice, first for 1.5 h and then for 1 h. Two of the boiled ingredients were filtered, mixed together, and concentrated in a 1 : 1 ratio (100% concentration) and stored at 4°C for later use. TXYF was diluted in distilled water to a concentration of 4.92 g/mL and stored at room temperature before use.
The following reagents were used: glutamine (ST083; Beyotime Biotechnology, China), PBS buffer (C0221A; Beyotime Biotechnology, China), and distilled water (GB19298; Watsons, China). The following instruments were used: linear accelerator (Elekta, Sweden), light microscope (Nikon, Japan), and histotome (LEICA, Germany).

HepG2 cells were grown in high glucose DMEM culture medium. All cell lines were cultured in medium supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), 1% mycillin, and 1% Glutamine (Beyotime, China) at 37°C in a humidified atmosphere with 5% CO₂. Using female BALB/c nude mice (4–6 weeks of age), HepG2 tumor model was established by subcutaneous injection of 2 × 10⁶ HepG2 tumor cells (0.1 mL) into the right upper flanks. When the tumor volume reached 0.8~1.0 cm in diameter (2-3 weeks after inoculation), the tumor-bearing mice were used for SPECT imaging and biodistribution studies. All animal studies were approved by Clinical Center at the FMMU.

OCI-LY3 and OCI-LY10 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Hyclone, USA) with, 10% fetal bovine serum (Gibco, USA), 1% penicillin/streptomycin (Beyotime, Shanghai), and 0.5% Glutamine (Beyotime, Shanghai) in 5% CO2 at 37°C. All cells were used within 20 passages.

NSCs were isolated and induced for differentiation as reported (41 (link), 42 (link)). Briefly, NSCs were prepared from the cerebral cortex of E14 Sprague–Dawley rats as described. The cells were maintained in DMEM/F12 medium (Cat. MA0214, Meilunbio) supplemented with 2% NeuroCult SM1 Neuronal Supplement (Cat. 05711, Stem Cell), 1% N2 Supplement-A (Cat. 07152, Stem Cell), 1% penicillin–streptomycin (Cat. C0222, Beyotime), 2 mM glutamine (Cat. C0212, Beyotime), 20 ng/ml recombinant bFGF (Cat. 450-33, Pepro Tech), and 20 ng/ml EGF (Cat. E9644, Sigma) on poly-l-lysine- (Sigma) and fibronectin- (Invitrogen) coated Petri dishes. EGF and bFGF were removed to induce NSC differentiation (43 (link), 44 (link), 45 (link), 46 (link), 47 (link)). Morphology of the cells was observed every 24 h during differentiation. From 24 h to 72 h after initiation of differentiation, the cells underwent significant morphological changes including the generation and elongation of the processes, suggesting that differentiation was successfully initiated.
COS7 cells were maintained and cultured in a DMEM medium (Cat. MA0212, Meilunbio) plus 10% FBS (Cat. S711-001S, Lonza). Cells were transfected using Lipofectamine 3000 (Cat. L3000015, Invitrogen) according to the supplier’s guidelines.
For MB-3 and MG132 treatments, the doses used were 50 μM for MB-3 (Sigma M2449) and 20 μM for MG132 (Selleck S2619).