Ab65359

Total cholesterol measurements of cell extracts were performed using a fluorometric cholesterol + cholesterol ester detection kit (Abcam ab65359). Triplicate sets of cells treated for 7 days as described above and samples were subjected to fluorometric total cholesterol detection according to manufacturer instructions.

Fasting levels of insulin and leptin were determined using rat/mouse insulin (Millipore, Catalog #EZRMI-13K) and mouse leptin (Millipore, Catalog # EZML-82K) ELISA kits, according to the manufacturers’ instructions, using plasma collected at euthanization. Plasma cholesterol was measured using a colorimetric cholesterol quantitation assay kit (Abcam, Catalog #ab65359), according to the manufacturer’s directions. Two APOE3 control and two APOE3 WD animals exhibited negative insulin concentrations and were excluded from insulin analyses. One APOE3 control animal exhibited a negative leptin concentration and was excluded from leptin analysis.

The fasting blood glucose level was measured by glucometer weekly through the tail prick method up to six weeks of the experiment. The serum glucose and insulin concentrations were estimated by a rat glucose assay kit (81693, crystal chem, Elk Grove Village, IL, USA) and an insulin ELIZA kit (ELR-insulin, RayBio^®, Peachtree Corners, GA, USA). Glycosylated hemoglobin was estimated using a rat hemoglobin Hb1Ac assay kit (MBS2033689, My BioSure, Grass Valley, CA, USA). The serum triglyceride, high-density lipid (HDL), low-density lipid (LDLs), and cholesterol levels were calculated using commercially available kits (ab65359, ab65336, and ab65390, respectively; Abcam, Cambridge, UK). The serum level of liver function enzymes, i.e., alanine aminotransaminase (ALT) and aspartate aminotransferase (AST), were determined through the kit assay method (ab105134/k752-100 and ab105135, respectively; Abcam, Cambridge, UK).

LDs were stained with a BODIPY 493/503 probe (25892‐10; AmyJet Scientific). After cell plating on glass coverslips and fixation, the cells were washed and incubated with 1 μg/mL BODIPY 493/503 for 10 min at room temperature. The number and area of the BODIPY dots per cell in at least 100 cells per independent experiment were determined with ImageJ software. Lipid accumulation in tumour tissue was visualized by oil‐red O staining. Tissue sections were fixed in 2% paraformaldehyde and stained with oil red O (C0158S; Beyotime) for 30 min at room temperature. Sections were washed with 60% isopropanol and water before imaging. Intracellular triglyceride (TG) and total cholesterol (TC) contents were quantified with a TG assay kit (ab65336; Abcam) and a TC assay kit (ab65359; Abcam) according to the manufacturer's instructions.

The total cholesterol, free cholesterol, and cholesteryl esters were extracted from the ileum (100 mg) by chloroform/isopropanol/NP-40 (7:11:0.1) and concentrations were determined using a commercially available fluorometric cholesterol/cholesteryl ester quantitative assay (Abcam, Ab65359) as per the manufacturer’s instructions.

Blood was collected from the heart via cardiac puncture, and serum was isolated after centrifuging at 1000g for 5 min. Aspartate aminotransferase (MAK055, Sigma-Aldrich), alanine transaminase (MAK052, Sigma-Aldrich), cholesterol (AB65359, Abcam), and TGs (MAK266, Sigma-Aldrich) were estimated according to the manufacturer protocol.