A112 1

All protocols were approved by the Institutional Animal Care and Use Committee (IACUC-1910021). Male Apoe^-/- mice on C57BL/6 background (The Jackson Laboratory) at 6-weeks old were randomly assigned into treatment or control group to receive i.p. injection of 2 mg/kg Romidepsin 17 (link), 18 (link) or the same volume of PBS once every 4 days until tissue harvest. Both groups were fed a high fat diet (Research Diet, D12108C) for 12 weeks. Mice were euthanized by CO₂ after overnight fasting. Blood was collected to measure serum total cholesterol (Nanjing Jiancheng, A111-1), triglyceride (Nanjing Jiancheng, A110-1), high density lipoprotein cholesterol (Nanjing Jiancheng, A112-1), low density lipoprotein cholesterol and glucose (Nanjing Jiancheng, F006-1-1). To assess lesion area, heart-aorta complexes were excised and thoracic-abdominal aortas fixed with 10% formalin, while aortic sinuses and arches embedded with optimal cutting temperature for frozen section preparation 19 (link).

The levels of total triglyceride (TG), total cholesterol (TC), high-density-lipoprotein cholesterol (HDLC), low-density-lipoprotein cholesterol (LDLC), and total bile acid (TBA) in serum were measured by analysis kits (A110-2, A111-2, A112-1, A113-1, and E003-2-1, Jiancheng Institute of Biotechnology, Nanjing, China). Non-esterified fatty acid (NEFA) was measured by an automatic biochemical analyzer (Olympus Au5400). The concentrations of very-low-density-lipoprotein-cholesterol (VLDL-C) and lysophosphatidylcholine (LPC) were measured by enzyme-linked immunosorbent assay kits (H249, Jiancheng, Nanjing and CEK621Ge, Cloud-clone corp, Wuhan, China, respectively) according to the instruction manual.

After treatment with RA-XII, HepG2 cells were harvested, washed with PBS, and lysed in 1% Triton X-100 lysis buffer (Beyotime, Nanjing, Jiangsu, China) for 30 minutes. Tumor tissues were weighted and homogenized at 2500 rpm/min for 10 min, then centrifuged, and the supernatants were harvested. Protein concentration was assessed by BCA kit (Thermo, Rockford, IL, USA). TC, TG, LDL and HDL in the HepG2 cell lysates or solid tumors were assayed using commercial kits (A111-1, A110-1, A113-1, A112-1, Jiancheng, Nanjing, China) according to manufacturer’s specifications. This study set blank wells, standard wells, and detected sample wells. The blank wells were added with sample diluent and other wells were added with standard preparation or detected samples, respectively. For TC and TG detection, all wells were then filled with working solution at 37 °C for 10 min and Optical density (OD) values were measured at 510 nm. For LDL and HDL detection, all wells were then filled with working solution A and working solution B at 37 °C for 5 min successively and OD values were measured at 546 nm two times.