Colo320

Normal colon cell line NCM-460 cells and human CRC cell lines CoLo205, DLD-1, HT-29, CoLo320, RKO, NCI-H716, and Caco-2 cells were from ATCC (Manassas, VA, USA). The NCM-460 cells were grown in M3 medium containing 10% FBS (Gibco, Grand Island, NY, USA). All of the CRC cell lines were grown in RPMI1640 supplemented with 10% FBS (Gibco) 37 °C/5% CO₂. For oxaliplatin (L-OHP) treatment, HT-29 and Caco-2 cells were treated with different doses of L-OHP (0, 5, 10, 20 and 40 μg/mL). For irinotecan (CPT-11) treatment, HT-29 and Caco-2 cells were treated with different doses of CPT-11 (0, 5, 10, 20 and 40 μg/mL). To study the protein stability, cycloheximide (CHX, 20 μg/mL) was administered for 0.25, 0.5, 1, 2, and 4 h. To block the ubiquitin–proteasome pathway, the cells were treated with 20 μM MG132. CHX and MG132 were purchased from MedChemExpress. shNC, sh-circ_RNF13-1, sh-circ_RNF13-2, sh-DDX27-1, sh-DDX-27-2, sh-TRIM24, sh-FBXW7, sh-MDM2, sh-TRIM25, sh-NEDD4, sh-TRIM11, sh-RNF180, sh-SYVN1, sh-MIB1, and pcDNA3.1-circ_RNF13 were purchased from GenePharma (Shanghai, China). HT-29 and Caco-2 cells were transfected 0.5 μg shRNA and/or plasmids using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). For double knockdown, 0.5 μg sh-circ_RNF13-1 and/or 0.5 μg sh-FBXW7 were co-transfected into CRC cells. At 48 h post-transfection, cells were harvested for subsequent analysis.

Human colorectal adenocarcinoma HT-29 cell line (ATCC) was used to collect conditioned medium for EVs isolation. We used human BRCA1-deficient fibroblasts, previously described [16 (link)], as target cells for exposure to EVs. Human colorectal adenocarcinoma Colo320 cell line (ATCC) was used as positive control for the validation of the antibodies used in this study. Murine colon adenocarcinoma MC38 cell line was given by Dr. Pnina Brodt. Cells were maintained in DMEM-F12 medium supplemented with 10% fetal bovine serum, and penicillin/streptomycin antibiotics (Wisent, Saint-Bruno, Canada). When BRCA1-KO fibroblasts reached 30% confluence, they were treated with complete DMEM-F12 medium supplemented with 30 μg/ml of EVs isolated from HT29 cells conditioned medium, cancer patient sera or control sera (as described below). Cells were maintained in these media at 37 °C in humidified atmosphere containing 95% air and 5% CO₂ with medium change every second day for 3 weeks. When cells reached 80–90% confluence, they were passaged 1 in 6 using 0.05% Trypsin-EDTA (Wisent, Saint-Bruno, Canada).

RKO, SW480, COLO320, HCT116 and HT29 human colon cancer cells (ATCC, Manasss, VA) were cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma, Saint Louis, MO), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Life Technologies) at 37°C in a humidified 5% CO₂ atmosphere.

Unless otherwise stated, chemicals were obtained from Sigma‐Aldrich (St. Louis, MO, USA), and cell culture reagents and cell growth medium from Gibco (Grand Island, NY, USA). Colorectal cancer cell lines (SW480, SW620, DLD‐1, HCT116, LS174T, T84, LOVO, Caco‐2, and Colo320) were from ATCC. DKO‐4, HKe3‐Parental (KRAS^WT/G13D−), HKe3‐KRAS wt (KRAS^WT/WT+), and HKe3‐KRAS mutant (KRAS^WT/G13D+) cell lines were provided by Senji Shirasawa [23 (link), 36 (link), 37 (link)]. Mycoplasma contamination tests were performed regularly, and cells with low passage numbers were used for the experiments. All CRC cell lines were maintained at 37 °C in humidified 5% CO₂ in air, and stock cells were maintained in RPMI‐1640 medium (Cat. No: 21875) containing 2 mml‐glutamine, supplemented with 10% FBS (Cat. No: 10270106; Gibco, Life Technologies).

Human cell lines used in this study (COLO320, RKO, HCT116, SW480, SW620, Geo, CaCo2, WiDr, HCT15, and HT29) were obtained from ATCC or The Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Germany). All cell lines were maintained in the medium recommended by ATCC in a humidified atmosphere with 5% CO₂ at 37 °C. As soon as they reached a confluence above 70%, cells were washed with PBS, trypsinized, and further cultivated.

SW480, COLO320, CaCo-2 and LS174T cell lines were purchased from ATCC. Upon receipt, cells were frozen, and individual aliquots were taken into cell culture, typically for analysis within 15 passages. Cells were grown in RPMI (SW480 and COLO320), DMEM (CaCo-2) or DMEM/F12 (LS174T) medium supplemented with 10% (SW480 and COLO320) or 15% (LS174T and CaCo-2) FBS and 1% penicillin/streptomycin. The stable SW480 cell line expressing GFP-TNKS1 was described earlier [22 (link)]. Testing for mycoplasma contamination was performed every sixth week. For inhibition of TNKS activity, cells were treated with 0.5 μM G007-LK for 6 h. DMSO was used as a control. For inhibition of proteasomal activity, cells were treated with 10 μM MG132, 25 nM Epoxomicin or 10 μM Lactacystin. Other inhibitors used were: 10 mM 3-Methyladenine (3-MA, autophagy inhibitor), or 300 μM Leupeptin (protease inhibitor) for indicated time points, either alone or in combination with G007-LK.