Puromycin

A lentivirus carrying the oxygen-dependent degradation domain-luciferase (ODD-Luc) fusion protein comprising HIF-1α (amino acid 530–652, a unique ODD) fused to the N-terminus of firefly luciferase was designed and produced by DesignGene Biotec, Co., Ltd. (Shanghai, China) (Additional file 1: Table S2). Jurkat cells were seeded at 10⁵ cells per well in 24-well plates and maintained in RPMI 1640 culture medium (Hyclone, USA) supplemented with 10% FBS (Gibco, USA). Lentivirus carrying ODD-Luc was added to the medium at a multiplicity of infection of 50 to transfect Jurkat cells for 3 days. Fresh culture medium was replenished, and the cells were then cultivated under standard culture conditions (37 ℃, 5% CO₂) for an additional 2 days. Finally, 5 µg/ml puromycin (Biosharp, China) was added to the culture to select stably transfected cells for subsequent use.
Lentiviruses carrying hypoxia-responsive elements-green fluorescent protein (HREs-GFP) were designed and produced by DesignGene Biotec (Additional file 1: Table S2). Similarly, the HREs-GFP reporter Jurkat cell line was generated by lentivirus transfection and subsequent puromycin selection, and stably transfected cells were prepared for subsequent use.

The lentiviruses packaging STC2 shRNA (targeting sequences: #sh1: GGTGAGCGAGGTAGCAAGA, #sh2: CACAGGTTCGGCTGCATAA) were purchased from Tsingke Biotechnology (Beijing, China). These sequences were cloned into the Plko.1-puro vector individually. Lentiviral packaging experiments were conducted using Lipofectamine 3000 (Invitrogen), as manufacture described. STC2 knockdown plasmid was co-transfected with the packaging plasmids (pMD2.G, psPAX2) into HEK293T cells. The lentivirus was collected 48 h after transfection. To generate stable STC2 knockdown cell lines, 143B and SJSA-1 osteosarcoma cells were transduced with lentiviruses and then selected with puromycin (2 µg/ml, Biosharp) after 2 weeks of production.

To construct TUSC5A and TUSC5B over-expression cell models, C3H10 T1/2 cells were infected with lentivirus carrying TUSC5A or TUSC5B in the presence of 8 ug/mL Polybrene (Biosharp, Guangzhou, China) and screened with 4 ug/mL puromycin (Biosharp, Guangzhou, China) for 5–7 days. Subsequently, over-expression of TUSC5A and TUSC5B was confirmed using qPCR and Western blot. Information of primers used for qPCR are shown in Table S2. 6His-tag antibody was used to detect overexpressed TUSC5A and TUSC5B proteins using Western blot. We used cop-GFP as a sorting condition to estimate the percentage of positive cells in the FL1 channel of the flow cytometer.

Imatinib-sensitive K562 cells were kindly given by Institute of Clinical Pharmacology (Central South University), imatinib-resistant K562/G01 cells were kindly given by Tianjin Institute of Hematology. K562 and K562/G01 cells were cultured in RPMI 1640 cell medium (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, South America) and 1% penicillin-streptomycin (Gibco, United States) mixture maintained at 37°C in a cell incubator with a humidified atmosphere containing 5% CO2. K562/G01 was cultured with 4 μM imatinib to keep its resistance, and removed imatinib treatment 1 week before experiment.
For hsa_circ_0058493 downregulation, K562/G01 cells were infected with lentivirus vector (GV493) carrying short hairpin RNA (shRNA) against hsa_circ_0058493 (sh-circ_0058493) and its negative control (sh-NC) (Genechem, Shanghai, China) following the manufacturer’s instructions. Puromycin (biosharp, China) were added into cells after transfection to screen successfully transfected cells. The sequence of sh-circ_0058493: sh-circ_0058493-1, CTC​ACC​AGG​GTT​CAG​CCG​T; sh-circ_0058493-2, TCA​CCA​GGG​TTC​AGC​CGT​C; sh-circ_0058493-3, TTT​ATT​CTC​ACC​AGG​GTT​C, and the one with the best knockdown efficiency would be chosen to conduct the subsequent experiments.

Lovastatin (purity ≥ 98.5%, HPLC) was from Shanghai Pharm Valley. Sodium mevalonate (#4667), anti-FLAG M2 agarose beads (#A2220), phenylmethanesulfonyl fluoride (PMSF, #P7626), protease inhibitor cocktail (#P8340), and β-mercaptoethanol (#M3148) were from Sigma-Aldrich. DiI-LDL was from Yeasen (#20614ES76). GW3965 (#10054) was from Cayman. Lipofectamine RNAiMAX (#13778150) was from ThermoFisher. SUMO1-AMC (#UL-704), ubiquitin-AFC (#U-551-050), and MG132 (#I-130) were from Boston Biochem. Puromycin (#BS111) was from Biosharp. G418 (#345810), pepstatin A (#516481), and ALLN (N-acetyl-leu-leu-norleucinal, #208719) were from Calbiochem. Ni-NTA Agarose (#30230) was from Qiagen. Linear polyethylenimine (#23966-1) was from Polysciences. FuGENE HD (#E2311) and M-MLV RTase (#M1701) were from Promega. Leupeptin (#11034626001) was from Roche. DL-Dithiothreitol (DTT, #A100281) and NP-40 (A100109) were from Sangon Biotech. Lipoprotein-deficient serum (density >1.215 g/ml) and delipidated-fetal calf serum were prepared in our laboratory as described previously (50 (link), 51 (link)). The purified PCSK9 protein was kindly provided by Dr Yan Wang (Wuhan University).

The GC cell lines MKN45 and AGS were bought from Procell (Wuhan, China). SGC7901 and BGC823 were bought from Beyotime (Shanghai, China). MGC803 and the gastric mucosal epithelial cell line GES-1 were purchased from iCell Bioscience (Shanghai, China). All cells were maintained under standard culture conditions using DMEM containing 10% FBS at 37 °C and 5% CO₂. All cells were checked for mycoplasma by a PCR-based method as well as 4′,6-diamidino-2-phenylindole (DAPI) staining to ensure the absence of contamination.
Cells were seeded in 6-well plates at a density of 2 × 10⁵ per well and cultured in a 37 °C incubator overnight. The shRNA plasmid (GenePharma, Suzhou, China) was transfected into the cells using Lipofectamine 3000 (Invitrogen, Los Angeles, CA, USA) in a serum-free medium. Cells were changed to complete medium at 6 h after transfection and cultured for another 30 h. Stable circMTA2 overexpression cell lines were established using lentivirus preparations infection and puromycin (Biosharp, Beijing, China) selection. The target sequences of shRNAs and the overexpression plasmid are provided in Supplementary Table S1.