E1015 105

Cells were seeded at 6 × 10⁶ cells/well in 6-well culture plates with triplicates for KO-ASIP bMECs and WT bMECs. These bMECs were digested with 0.25% trypsin, the cell suspension was collected with a 1.5 mL clean centrifuge tube, and cells were collected by centrifugation at 700 r. It was washed again with PBS. Add Lysis Buffer for Triglyceride or Cholesterol Extraction. Triglycerides and CHOL were measured using a TG detection kit and a tissue/cell total CHOL assay kit (E1015-105 and E1013, Applygen Technologies, Beijing, China), respectively. The cellular contents of TG and CHOL were normalized by protein content. The concentrations were measured using a microplate reader (SM600, Shanghai YongChuang Medical Instrument Co., Ltd., Shanghai, China). Cells were collected at a cell volume of 50 mg per tube, three tubes each from KO-ASIP bMECs and WT bMECs and sent to Shanghai Applied Protein Technology Inc to determine the intracellular fatty acid content.

For quantitative estimation of triglycerides or phospholipids in CRC cells, an enzymatic assay was performed using EnzyChrom Triglyceride Assay Kits (BioAssay Systems, Hayward, CA) and EnzyChrom Phospholipid Assay Kits (BioAssay Systems, Hayward, CA) according to the manufacturer's protocols. Cellular cholesterols were assayed using a total tissue cholesterol kit (E1015-105, Applygen Technologies, Beijing, China).