Opti mem 1 1

KEAP1-depleted mouse embryonic fibroblasts (MEFs^Keap1−/−) were kind gifts from Thomas Kensler (University of Pittsburgh, Pittsburgh, PA, USA) [67 (link)]. Media, serum, and supplements for cell culture were purchased from Sigma-Aldrich, Biowest, and Corning, respectively. MEFS were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) plus 10% heat-inactivated fetal bovine serum and 1% Penicillin/Streptomycin/L-Glutamine (P/S/G, Corning Product, Corning, NY, USA). OPTI-MEM I (1×) (Gibco) was used for transient transfections.

HeLa cells (0.8 × 10⁵ cells/well) were seeded on coverslips (12 mm Φ; Microscope Cover Glasses; no. 0111520) placed in a 24-well plate. Following 12 hours, cells were washed two times with pre-warmed (37°C) Dulbecco’s PBS (Biological Industries). The buffer was replaced with 450 µL of DMEM without antibiotics ([DMEM-High-Glucose, SARTORIUS] supplemented with 10% fetal bovine serum [FBS] [Qualified FBS, Gibco] and 1% GLN [L-glutamine, SARTORIUS]). A “transfection mixture” was prepared by adding 500 ng of the relevant plasmid to 50 µL of Opti-MEM I (1×) (Gibco, no. 31985-047), pipetting up and down 10 times followed by the addition of 1.5 µL of TransIt-X2 solution (MIRUS, no. MIR 6004). The “transfection mixture” was then mixed again, incubated at 22°C for 20 minutes, and then added to the cells in 450 µL DMEM complete media without antibiotics. Cells were then incubated for 4–5 hours in the CO₂ incubator (37°C, 5% CO₂, 95% humidity). Thereafter, the medium was aspirated and replaced with a fresh growth medium. Cells were analyzed ~24 hours after transfection.

Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen, 11668-019) according to the manufacturer’s instructions. Briefly, a dilution of plasmid and Lipofectamine was made using OPTI-MEM I 1× (GIBCO, cat. no. 31985-047) and then added to the cells. After 4 h, the medium was removed and fresh complete DMEM medium was added. Cells were allowed to grow for 48 h before analysis.

SW480 cells were obtained from the Chinese Academy of Sciences SUER0200 (XR). RPMI-1640 culture media (1×) was provided by KGI bio-KGM41500S-500; trypsin-EDTA digestive juice, crystal violet staining solution and Annexin V-FITC/PI, and apoptosis kit were purchased from Solarbio T1300. PBS was obtained from BI 02-024-1ACS, and OPTI-MEM® I (1×) was purchased from gibco 331985-062. TGF-β1 was provided by Beijing Boaosen Biotechnology Co., Ltd. AG12051847, CCK8 cell proliferation detection reagent was obtained from KGI Bio-KGA317, and PPTS was extracted from the plant Pulsatilla [18 (link)].