Lox1 sirna

For cell experiments of LOX1 and STRA6 knockdown, HK-2 cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well in 2 ml antibiotic-free medium and then cells were cultured at 37°C and 5% CO₂ until the cell growth covered 80% of the area of the dish. After overnight incubation, negative control scramble siRNA (Santa Cruz Biotechnology Inc.), LOX1 siRNA (Santa Cruz Biotechnology Inc.) or STRA6 siRNA (Santa Cruz Biotechnology Inc.) was mixed into transfection reagent (Santa Cruz Biotechnology Inc.). The transfection medium (Santa Cruz Biotechnology Inc.), and the mixture were added into cells and incubated for 7 h. These cells were placed in fresh medium for 24 h, and then stimulated with PBS, native L1 (50 μg/ml), or L5 (50 μg/ml) for 24 h.

The pCMV6‐XL5‐LOX‐1 and SIRT1 plasmids, from Origene Technologies (Rockville, MD, USA), were constructed with full‐length human LOX‐1 cDNA OR SIRT1 and transfected into HUVECs with a FuGene 6 transfection reagent (Roche Diagnostics, Mannheim, Germany).18 Empty vectors were transfected as controls. After 48 hours transfection, HUVECs were incubated with P‐II and HHcy. Then, cells were collected for analysis.
For siRNA experiments, HUVECs were transfected with LOX‐1 siRNA or SIRT1 siRNA (Santa Cruz Biotechnology). Briefly, HUVECs were cultured in antibiotic‐free Dulbecco's modified Eagle's medium at 37°C for 24 hours, then the siRNA duplex solution was added. Cells were subjected to each experiment 24 hours after transfection.