Hek293t cells

HEK293T cells were obtained from American Type Culture Collection (ATCC, #CRL-11268, RRID:CVCL_1926) and grown in heat-inactivated Dulbecco’s modified Eagle’s minimal essential medium (DMEM, Thermo Fisher Scientific, Waltham, MA, US, #41966-029) supplemented with 10% heat inactivated foetal bovine serum (FBS, Thermo Fisher Scientific, #26140079) and 1% PenStrep (Thermo Fisher Scientific, #15140122) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Transient transfection of plasmids encoding EV, rCP-WT, and rCP mutants into HEK293T cells was performed using Lipofectamine 2000 (Thermo Fisher Scientific, #11668-019) and Opti-Mem (Thermo Fisher Scientific, #31985062) according to the manufacturer’s instructions. Media were supplemented with 10 μM CuSO₄·5H₂O 5 h and 21 h post-transfection, with the exception of the WT negative control. 48 h after transfection, 4 mL of each collected cell supernatant were concentrated and buffer-changed to Tris buffer (20 mM, pH 7.0) using Amicon-Ultra-4 30 kDa devices (Merck, Darmstadt, DE, #UFC8030). Transfected cells were lysed with M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, #78501) supplemented with a Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, #87786). The total protein concentration in each cell lysate or supernatant was determined using the Bradford assay (Thermo Fisher Scientific, #23236).

Human differentiated colorectal adenocarcinoma HT29 cells, human normal intestinal epithelial HIEC‐6 cells, and HEK293T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HT29 and HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin and penicillin (Gibco, Grand Island, NY, USA) at 37 °C in 5% CO2. RPMI 1640 medium supplemented with 10% FBS, streptomycin, and penicillin (Gibco) was used for HIEC‐6 cell culture.
Hydrogen peroxide (H₂O₂) was purchased from Sinopharm Chemical Reagent Co. Ltd. The USP7 inhibitor P5091, proteasomal inhibitor MG132, and protein synthesis inhibitor cycloheximide were purchased from Selleck Chemicals and Sigma‒Aldrich (St. Louis, MO, USA). The antibodies used in this study were as follows: anti‐AMBRA1 (13762‐1‐AP, Proteintech (Rosemont, IL, USA), 1:1000 for WB, 1:500 for IP and 1:100 for immunohistochemistry [IHC]), anti‐NRF2 (16396‐1‐AP, Proteintech, 1:1000 for WB, 1:100 for IHC and IF), anti‐HO‐1 (10701‐1‐AP, Proteintech, 1:1000 for WB and 1:100 for IHC), anti‐NQO‐1 (11451‐1‐AP, Proteintech, 1:1000 for WB and 1:100 for IHC), anti‐DUB3 (26143‐1‐AP, Proteintech, 1:1000 for WB), anti‐USP7 (66514‐1‐Ig, Proteintech, 1:1000 for WB), anti‐HA (3724, Cell Signaling Technology [Beverly, MA, USA], 1:1000 for WB), anti‐FLAG (14 793, Cell Signaling Technology, 1:1000 for WB), anti‐MYC (2276, Cell Signaling Technology, 1:1000 for WB), anti‐β‐actin (A2228, Sigma‒Aldrich, 1:10000 for WB) and anti‐Lamin A (10298‐1‐AP, Sigma‒Aldrich, 1:10000 for WB). Normal rabbit IgG (2729; Cell Signaling Technology) was used.
The pRK7‐FLAG‐AMBRA1, pcDNA‐HA‐AMBRA1, pcDNA‐MYC‐AMBRA1, pRK7‐FLAG‐KEAP1, pCDNA‐GFP‐NRF2, pcDNA‐HA‐NRF2, pRK7‐FLAG‐DUB3, pcDNA‐HA‐DUB3, pRK7‐FLAG‐USP7, pcDNA‐HA‐USP7, and pcDNA‐MYC‐USP7 vectors were constructed using the ClonExpress I One Step Cloning Kit (Vazyme, Nanjing, China). HA‐Ub, HA‐Ub‐K48R, and HA‐Ub‐K63R plasmids were kindly provided by Dr. Wei Yu of Fudan University. Expression plasmids for mutant AMBRA1, KEAP1, and USP7 were generated using a KOD mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.