Ox ldl

RASMCs seeded to 6 or 12-well dishes were transfected by siRNA targeting SERPINA3 and scramble siRNA (siCtr) (Genepharma) at about 70–80% confluency, by using Lipofectamine® RNAiMAX reagent (Invitrogen) according to the user manual. After 1 day, transfected cells were stimulated by ox-LDL (50 μg/mL, Cat#sc-7950002, Santa Cruz) for 12 h. HUVECs were stimulated with ox-LDL (100 μg/mL) and human recombinant SERPINA3 protein (the following text is written as SERPINA3, 100 ng/mL, Cat#ag2830, Proteintech) for 18 h. All cells were then used for the following experiments.

Peritoneal macrophages were obtained by injecting 3 mL of 4% Brewer’s thioglycollate (Merck) intraperitoneally followed by a collection of the elicited peritoneal exudate cells for 4 days after injection. Exudate cells were centrifuged to use as ex vivo samples or resuspended in RPMI medium (Life Technologies, Carlsbad, CA, USA) with 100 U/mL of penicillin and 100 μg/mL of streptomycin (Nacalai Tesque, Kyoto, Japan) and 10% fetal bovine serum (Lonza, Walkersville, MD, USA) at 37 °C with 5% CO₂. The cells were then incubated with or without 40 μg/mL of ox-LDL (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 24 h. In KD examination, AT1R, ELAVL1, PPARγ, ABCA1, and control siRNA (Thermo Fisher Scientific) were transfected with lipofectamine (Thermo Fisher Scientific) and the mRNAs of interest were analyzed by real-time PCR after 24 h. For the scratch assay, cells were grown to confluence on 24-well tissue dishes, and a single scratch was made using a sterile 1000-μl pipette tip. Photographs were taken after 12 h, and the cell-covered area was measured by Image J. In overexpression examination, ABCA1 cDNA clones (Santa Cruz Biotechnology Inc.) were transfected with UltraCruz Transfection Reagent (Santa Cruz Biotechnology Inc.) for 24 h, and then ox-LDL were added. The mRNAs of interest were analyzed by real-time PCR after 24 h.