Sw480 ccl 228

Human CRC cell lines SW480 (CCL-228) and SW620 (CCL-227) were from ATCC (Manassas, VA, USA). Cells were initially cultured in 175-cm² flasks in RPMI-1640 medium (Life Technologies, CA) supplemented with 10% (v/v) foetal bovine serum (Thermo Fisher Scientific, CA) and 1% (v/v) Penicillin Streptomycin (Pen/Strep, Thermo Fisher Scientific) at 37°C and 10% CO₂ and then transferred to CELLine AD-1000 Bioreactor Flasks (Integra Biosciences, NH) and continuous culture performed over a period of several weeks, as described [26 (link)]. Cell culture medium (CM) from two separate bioreactor flasks (150 ml) was collected for EV isolation (in duplicate, biological replicates), as described [26 (link)]. Briefly, low-speed centrifugation removed cells and cell debris. Then the supernatant was centrifuged at 10,000 × g for 30 min to obtain shed microvesicle (sMVs). The sMV-depleted supernatant was further centrifuged at 100,000 × g for 1 h to isolate crude exosomes. OptiPrep density gradient (100,000 × g 18 h) was employed to obtain purified exosomes. Protein quantification was performed by protein staining (SYPRO Ruby) densitometry following 1D-SDS-PAGE [26 (link)].

Human CRC cell lines, HCT116 (CCL-247) and SW480 (CCL-228), were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The normal colon cell line, NCM460 (CC-Y1550), was procured from EK-Bioscience Biotechnology Co., Ltd. (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and cultured at 37℃ with 5% CO₂ in a humidified atmosphere. Short hairpin (sh) RNAs targeting KDM5C and PFDN5 were designed and synthesized by GenePharma Co., Ltd. (Shanghai, China). These shRNAs were cloned to interference vector pGPU6/GFP/Neo, respectively. Lentiviral vectors were generated by co-transfecting the interference agent with VSVG and D8.9 packaging vectors into 293 T cells (CRL-3216; ATCC, Manassas, USA) using Lipofectamine 3000. After 48 h, lentiviral vectors were collected and used to infect the HCT116 and SW480 cells. The infected cells were incubated for an additional 48 h under 5% CO₂ at 37 ℃ to establish cell lines with stable inhibition of KDM5C or co-inhibition of KDM5C and PFDN5. To rule out the potential impact of autophagy on apoptosis, cells with stable KDM5C inhibition were treated with 10 nM chloroquine (CQ, Sigma-Aldrich, USA) for 12 h prior to apoptosis detection using flow cytometry.