Dmem high glucose medium

Human pancreatic cancer cell lines (PANC-1, BXPC3, ASPC1) and the human pancreatic ductal epithelial cell line (HPDE6-C7) were obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM high-glucose medium (Biological Industries: 01-052-1A) containing 10% FBS (Biological Industries: 04-007-1A), 100 units/mL penicillin and 100 μg/mL streptomycin (HyClone, Logan, UT, USA). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂.

H9C2 and HEK-293T cells were purchased from the National Collection of Authenticated Cell Cultures, Chinese Academy of Sciences (China); they were cultured in DMEM (high glucose) medium (01-055-1A, Biological Industries, Israel) supplemented with 10% foetal bovine serum (04-010-1A, Biological Industries, Israel). The cells were kept at 37 °C in 5% CO₂ at atmospheric pressure.
Male Sprague Dawley rats (250 ± 10 g, 8 weeks old) were purchased from the Model Animal Research Center of Nantong University. The rats were kept in a specific pathogen-free (SPF) environment at 22 ± 1 °C, a relative humidity of 50 ± 1%, and a regular 12 h day-night cycle. All animal experiments were approved by the Medical Ethics Committee of Nantong University and conducted under the guidance of the Laboratory of Nantong University. This study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University (No. S20200314-012).

MDBK cells were purchased from China Center for Type Culture Collection (CCTCC). DMEM/F12(HAM) medium, DMEM low glucose medium, and DMEM high glucose medium were purchased from Biological Industries, Israel. LPS from Escherichia coli 055: B5 was purchased from Solarbio, China. Notch3 inhibitor (DAPT) was purchased from Selleck, America.

Human hepatocellular carcinoma cell lines HepG2, SMMC-7721, Huh7 and human normal liver cell line L02 were purchased from American Type Culture Collection (ATCC). Cells were maintained in Dulbecco's Modified Eagle medium (DMEM) high glucose medium (Hyclone, Logan, USA) with 5% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel) and grown in complete medium (DMEM high glucose medium with 10% FBS) at 37°C and 5% CO₂.

HEK293T cells (Meisen CTCC) were cultured in a DMEM/high glucose medium (Biological Industries, 01-052-1A) with 10% FBS (VISTECH, SE100-B) at 37℃ under 5% CO₂. The CpOGA^CD and CpOGA^DM sequences were codon optimized to Homo sapiens and Drosophila using Jcat (Grote et al., 2005 (link)). The fragments of TurboID-CpOGA^CD and TurboID-CpOGA^DM (TurboID-CpOGA^CD/DM) were PCR amplified and cloned into pCDH-CMV-HA vectors, respectively. For lentivirus preparation, HEK293T cells were transfected with TurboID-CpOGA^CD/DM plasmid with the packaging plasmids pPAX2 and pMD.2G using Polyethylenimine Linear (PEI, Polysciences, 24765). The PEI-containing medium was replaced with fresh serum-containing DMEM medium after 8 hr, and the viral supernatants were collected 48 hr and 72 hr post-transfection. The viral supernatants were centrifuged at 10,000 g for 1 hr at 4℃, and the pellet was dissolved in PBS (Biological Industries, 02-023-1A). HEK293T cells were infected in six-well plates and selected with 1 µg/mL Puromycin (Selleck, s7417) in the medium for at least 5 days. For biotin labeling, the TurboID-CpOGA^CD or TurboID-CpOGA^DM expressing HEK293T cells were labeled with 10–100 µM biotin (Merck, B4501) in the medium for 15 min to 3 hr. Labeling was stopped by placing cells on ice and washing cells three times with PBS (Biological Industries, 02-023-1A).

The cell line WRL68 was purchased from Mingzhou Biotechnology. Other cell lines were obtained from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences. Huh7, HepG2, A549, HeLa, K562, MCF7 and HEK293T cells were cultured in DMEM high glucose medium (Biological Industries, 0023119), and T24 and WRL68 cells were cultured in RPMI 1640 medium (Biological Industries, 2035127) with 10% fetal bovine serum (FBS) (Biological Industries, 1841924) in 5% CO₂ at 37 °C. All cells were identified without mycoplasma infection.
To overexpress genes in cultured cells, expression plasmids were stably transfected with a retrovirus-mediated transfection method. For this, HEK293T cells were transfected with pLVX-puro (empty plasmid) or pLVX-KHK-C-puro and the two packaging plasmids, psPAX.2 and pMD2.G. Two days later, the virus particles were collected, filtered, and used to infect the target cells. After 48 h of infection, transfected cells were selected with 2 μg/mL puromycin. Cells stably transfected with pLVX-puro were used as a control cell line.

Leukemia cell lines Nalm6, CCRF-CEM, Toledo, and K562 were kindly provided by Steve Feldman, and grown in standard culture conditions, using RPMI medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamate, Sodium Pyruvate, Hepes buffer 0.1 M (all from Biological Industries), and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich) (“target cell medium”). For activation and transduction, T cells were cultured in RPMI supplemented with 10% FBS, 2 mM L-Glu, 100 U/mL penicillin, and 100 μg/mL streptomycin and interleukin-2 (IL-2, 100 IU/ml, Novartis Proleukin) (“T cell medium”). 293T cells used for viral production were cultivated in DMEM high glucose medium supplemented with 10% FBS, 2 mM L-Glutamate, Sodium Pyruvate, Hepes buffer 0.1 M, and non-essential amino acids solution (all from Biological Industries). Alternatively, medium can be supplemented with human AB serum instead of FBS.