Rpmi 1640

The HGC-27, MKN-45, AGS cell lines were purchased from Runke Biotechnology (Guangzhou, China) and the MGC803 and HEK-293T were from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal gastric mucosa epithelial cell line GES-1 was purchased from Gefan Biological Technology (Shanghai, China). The MGC-803 and HEK-293T were cultured in high-glucose DMEM (Bioind, Israel). The HGC-27, MKN-45 and GES-1 cells were maintained in RPMI-1640 (Bioind, Israel) and the AGS cells were propagated in DMEM/F-12 Medium (Bioind, Israel). All cell lines were supplemented with 10% fetal bovine serum (FBS; Excell, China) in a humid environment with 5% CO₂ at 37 °C. The plasmid of hsa_circ_000200 (OE) was synthesized by BersinBio (Guangzhou, China), and ASO targeting hsa_circ_000200 was synthesized by RiboBio (Guangzhou, China). The sequence of ASO was TGTGTTGAAGGGACTGTTTA. Furthermore, miR-4659a/b-3p mimics or inhibitors were designed and synthesized by GenePharma (Suzhou, China). The plasmids, ASO and miRNA mimics or inhibitors were transfected into cells with Lipofectamine 2000 (Invitrogen, USA). RNA collection and cell function experiments were performed 48 h after transfection. Protein collection was performed 72 h after transfection.

NCM460, HCT8, HT29, SW480, SW620, HCT116 and RKO CRC cell lines were purchased from the Chinese Academy of Science Cell Bank and cultured in DMEM and RPMI-1640 medium (BioInd, Beit Haemek, Israel) including 10% fetal bovine serum (FBS, BioInd), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco, Grand Island, USA) in a humidified atmosphere of 5% CO₂ at 37 °C.

A tetracycline (TET)-regulated TrkB-expressing NB cell line TB3 was utilized. In the presence of TET, TrkB expression is suppressed, and in the absence of TET, TrkB expression is induced [9 (link)]. TB3 cells were cultured in RPMI 1640 (Bioind, Israel) containing 10 % fetal bovine serum (FBS; Bioind, Israel), 2 mM glutamine, antibiotics, and puromycin (0.5 μg/ml), and in the presence or absence of tetracycline (1 μg/ml) at 37 °С in 5 % CO₂ incubator.

Ca(NO₃)₂, Na₂HPO₄, NaOH, polyethylene glycol-400 (PEG-400) and polyethyleneimine (PEI, M.W. 70,000) were purchased from Aladdin (Shanghai, China). We purchased 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Trypsin-EDTA solution, Lysotracker^TM Red and the QuantiChrom^TM calcium ion detection kit from Beyotime Institute of Biotechnology (Shanghai, China). High glucose Dulbecco’s modified eagle medium (DMEM) and Roswell park memorial institute-1640 (RPMI-1640) were obtained from BioInd (Beit-Haemek, Israel). Fetal bovine serum (FBS) was purchased from Gibco (Sidney, Australia). Fluorescently labeled siRNA-FAM, negative control siRNA (siNC, sense strand, 5′-UUC UCC GAA CGU GUC ACG UTT-3′), KRAS siRNA (siKras, sense strand, 5′-CAC CAU UAU AGA GAA CAA ATT-3′), and commercial transfection reagent siRNA-Mate were obtained from Genpharm (Shanghai, China). Other chemicals and reagents were of analytical grade.
Human pancreatic cancer cells PANC-1, BXPC-3, CFPAC-1, and human pancreatic ductal epithelial cell HPDE6-C7 were purchased from the cell bank of Chinese Academy of Science (Shanghai, China). PANC-1 and CFPAC-1 were grown in high glucose DMEM, and HPDE6-C7 were maintained in RPMI-1640 medium. Cells were incubated with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ atmosphere.

All the human cancer cell lines were obtained from ATCC. Cells were cultured in RPMI-1640 (BioInd) medium according to the instructions from ATCC, with the medium containing 10% FBS (BioInd), 1% antibiotics (penicillin and streptomycin) at 37 °C in an atmosphere of 5% CO₂.

Cal-27 cells, a human oral squamous cell carcinoma (OSCC) line with a key mutation in the autophagy protein BECN1 (Supplement Table), were kindly provided by Dr Shi Songtao, Shanghai Ninth People’s Hospital, China. The following cell lines were obtained from ATCC (Rockville, MD, USA): SCC-25, SCC-15 and Fadu (human OSCC cell lines); A2780 (a human ovarian cancer cell line); TE-1 (a human oesophageal cancer cell line); and MCF-7 (a human breast cancer cell line). A2780, TE-1 and MCF-7 cells have mutations in p62 (Supplement Table) (ref. ^{33 (link)}). The mutation site in MCF-7 cells is located in the PB1 domain, and the mutation site in A2780 cells is located in the UBA domain (Supplement Table). Cal-27, SCC-25 and Fadu cells were cultured in H-DMEM (Bioind, Kibbutz Beit HaEmek, Israel). A2780, MCF-7 and TE-1 cells were cultured in RPMI-1640 (Bioind, Kibbutz Beit HaEmek, Israel). SCC-15 cells were cultured in Ham’s F12 medium (HyClone, Utah, USA). All media were supplemented with 10% foetal bovine serum (FBS) (Bioind, Guangzhou, Guangdong, China) and 1% penicillin/streptomycin (HyClone). During starvation induction, cells were cultured in Earle’s balanced salt solution (EBSS) (HyClone). All cells were incubated in a thermostatic incubator (SANYO, Osaka, Japan) at 37 °C in 5% CO₂.