Short hairpin RNA (shRNA) transfection

The pLKO.1-GFP-LXRα shRNA Plasmids were purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-38829-SH). The transfected shRNA plasmid is a pool of 3 target-specific lentiviral vector plasmids. The three shRNA sequences are: Sequence #1, 5′-CCGGGATCTGGGATGTGCACGAATGCTCGAGCATTCGTGCACATCCCAGATCTTTTTTG-3′; sequence #2, 5′-CCGGAGTTCTCCAGGGCCATGAATGCTCGAGCATTCATGGCCCTGGAGAACTTTTTTTG-3′; and sequence #3, 5′-CCGGGCAACTCAATGATGCCGAGTTCTCGAGAACTCGGCATCATTGAGTTGCTTTTT-3′. The control pLKO.1-GFP-shRNA plasmid encodes a scrambled shRNA sequence that will not lead to the specific degradation of any cellular message. shRNA transfection was performed using the Lipofectamine^® 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, transfection was performed in a six-well tissue culture plate, at a density of 6×10⁵ cells with 50–70% confluency in antibiotic-free normal growth medium supplemented with FBS. Plasmid (1 µg) was transfected into 4×10⁵ cells/well. Subsequently, 72 h later, the cells were collected for downstream assays. The transfection efficiency was determined by RT-qPCR.