Penicillin streptomycin

MDA-MB-231 cells were obtained from Cell Bank, Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and maintained in DMEM medium (Gibco, Cat. No.:11965–092) supplemented with 10% fetal bovine serum (FBS, Gibco, Cat. No.: 10099–141) and 1% penicillin & streptomycin (Meilunbio, Cat. No.:MA0110) in a humidified incubator containing 5% CO₂ at 37 °C. MGC803 and HCT8 cells, also obtained from Cell Bank, Type Culture Collection of Chinese Academy of Sciences, were both cultured in RPMI 1640 medium (Meilunbio, Cat. No.: MA 0215). MCF7, gifted by Prof. Tu Hong from Shanghai Jiao Tong University (China), was maintained in DMEM medium supplemented with 10% FBS and 1% penicillin & streptomycin. MCF-10A cells and BT549 cells, obtained from Zhongqiao Xinzhou Biotechnology (Shanghai, China), were cultured in special medium (Cat. No.: ZQ-1311, Zhongqiao Xinzhou Biotechnology) and RPMI 1640 medium, respectively, supplemented with 10% FBS and 1% penicillin & streptomycin.

Human pancreatic cancer cell lines (BxPC-3, PANC-1, AsPC-1, and Capan-1) were obtained from the American Type Culture Collection (ATCC). MIA PaCa-2, SW1990, and the normal human pancreatic ductal cell line hTERT-HPNE were purchased from the Cell Bank of the Chinese Academy of Sciences. All cells were cultured in Dulbecco’s modified Eagle medium (DMEM: Gibco; Hyclone; Meilunbio) containing 10% fetal bovine serum (FBS: Gibco) and 1% penicillin/streptomycin (Meilunbio) at 37°C in an atmosphere with 5% CO2.

Cell lines of wild-type (WT) HepG2 and Nrf2^−/− HepG2 were supported by China Cell Line Bank (Beijing, China) and Dr. Ci Xinxin (Institute of Translational Medicine, The First Hospital, Jilin University), respectively. These cells were grown in 4.5 g/L glucose DMEM (Gibco, NY, USA), 10% fetal bovine serum (FBS, Clark, Australia), and 1% penicillin/streptomycin (Meilun Biotechnology, Dalian, China) in a humidified 5% CO₂/95% air atmosphere at 37°C. These cells were pretreated with 4.5 g/L glucose DMEM (serum free) for 3 h before treatment.
WT HepG2 cells were exposed to 1 mM FFA (oleate : palmitate = 2 : 1) for different time points from 0 h to 24 h. WT HepG2 cells were incubated in 0, 4, 20, 100, 200, and 500 μM GPS for 24 h. The cells were cultured with GPS for 1 h; after that, the cells were coincubated with 1 mM mixture FFA for 24 h. Cytotoxicity of FFA and GPS was assayed using CCK-8 kits (Invigentech, CA, USA).

HEK293T and K562 cells were cultured in DMEM (high glucose) medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin (Meilunbio, Dalian, China). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. For transfection, we co-transfected 2 ug of the editor plasmid and 1 ug sgRNA into HEK293T cells using poly-ethyleneimine (PEI, Polyscience, Warrington, PA, USA) following the manufacturer’s protocols in 12-well plates. Positive cells were isolated by flow cytometry 48 h after transfection, and genomic DNA was extracted using the Crude DNA Extraction Kit (catalog number P072, Vazyme, Nanjing, China). The genotyping of transfected cells was determined using gene-specific primers through nested PCR. In the first round of PCR amplification, Extaq (Takara, Shiga, Japan) was activated at 95 °C for 3 min, then for 30 cycles, with denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, with an extension at 72 °C for 1 min and a final extension at 72 °C for 5 min after the cycles. The second round of PCR was performed with inner nested primers through the same PCR program. The PCR products were purified for Sanger sequencing or next-generation sequencing. The genotyping identification primers used in this study are listed (Table S7). Next-generation sequencing data were analyzed using CRISPResso2 (version 2.2.7).

Primary NSCs were obtained from embryonic hippocampus on embryonic day 14.5. Briefly, the extracted tissue was digested for 10 min by trypsin and then gently dispersed. The suspension was filtered through a 40 mm filter and then collected. The hippocampus‐derived NSCs were cultured in suspension and maintained proliferation in DMEM/F12 (#11330‐032, Gibco) containing 20 ng mL⁻¹ EGF (#AF‐450‐33, Peprotech), 20 ng mL⁻¹ bFGF (#315‐09, Peprotech), 1% glutaMAX (Gibco), 1% B27(#17504‐044, Gibco) and 1% penicillin/streptomycin (Meilunbio). After 4–5 days, neurospheres occurred and were dissociated into single cells by accutase (#A11105‐01, Gibco) and then plated on plates coated with poly‐l‐ornithine (Sigma) and laminin (L2020, Sigma) in growth media.